CN101421609A - The method relevant, potpourri, kit and composition with analyte determination - Google Patents

Abstract

The present invention relates to and carry out the relevant method of analyte determination, potpourri, kit and composition by mass spectroscopy.

Description

The method relevant, potpourri, kit and composition with analyte determination

The cross reference of related application

The application requires in the U. S. application No.11/625 of submission on January 22nd, 2007,688 right of priority, and require in the U.S. Provisional Patent Application No.60/761 of submission on January 24th, 2006,711 and the U.S. Provisional Patent Application No.60/764 that submits on February 1st, 2006,216 rights and interests, all these applications all are incorporated herein by reference.

Each several part title used herein only for the purpose of tissue, and should not be interpreted as by any way described theme being limited.

Technical field

The present invention relates to and carry out the relevant method of analyte determination, potpourri, kit and composition by mass spectroscopy.

Introduction

The present invention relates to by quality analysis determination and analysis thing.Analyte can be interested any molecule.The non-limiting example of analyte includes but not limited to that protein, peptide, nucleic acid, carbohydrates, lipid, steroids and quality are less than 1500 daltonian micromolecule.Can utilize unique labelled reagent that analyte is measured, described labelled reagent makes can carry out relative quantification and/or absolute quantitation to the analyte in the complex mixture.Usage flag reagent is used for the sample mixture of Analysis of Complex in groups, and wherein labelled reagent can be isomeric (isomeric) and/or isobaric (isobaric).

With reference to Fig. 1 a, labelled reagent comprises report thing part, counterbalance (or connector) part and reaction active groups, and wherein said reaction active groups is combined in the thing that the analyte of response analysis thing form replaces.The example of the labelled reagent of this general formula and the analyte of mark has been disclosed among for example disclosed common unsettled and total U.S. Patent application US 2004-0219685 A1, US2005-0114042 A1, US 2005-0147982 A1, US 2005-0147985 A1, US2005-0147987 A1, US 2005-0148771 A1, US 2005-0148773 A1 and the US2005-0148774 A1.As what in the laid-open U.S. Patents application of being quoted, discussed, isomerism and/or isobaric labelled reagent group can be used for for example analyte of two or more different samples of mark, wherein the different labelled reagents in the group all have identical gross mass, but each report thing part can be encoded especially, makes each report thing part of this group all have unique quality.Because all having identical gross mass, all reagent in this group can comprise report thing part with unique qualities, so counterbalance (or connector) part also can (but and nonessential) comprise one or more heavy atom isotopes usually, with the quality of this " balance " each unique report thing, thereby the report of every kind of labelled reagent thing in this group/connector combination all has identical gross mass.

In Fig. 1 b, illustrate the example (as describing in detail) of new labelled reagent (or analyte of mark) herein.Although be not replace form (except R ₁And R ₂) set forth, but be to be understood that described labelled reagent can be that replace or unsubstituted.In diagram, some key is shown as fracture, thereby discharges unique report thing part and common but unnecessary counterbalance part at least from the analyte of labelled reagent or mark.For the analyte of mark, observed every kind of special report thing ion (sometimes being called signal ion (signature ion)) in mass spectrometer can be used for the amount of quantitative sample and/or sample mixture analyte then.

Fig. 2 a and 2b illustrate 9 kinds of different coding forms (R for example of the foundation structure shown in Fig. 1 b ₁And R ₂Can be hydrogen or methyl independently of one another), it can be used for promoting at least 9 heavily experiments (9-plex experiment), wherein asterisk ( ^*) be used to show in due course herein ¹²C atom quilt ¹³The C atom substitutes, perhaps ¹⁴N atom quilt ¹⁵The N atom substitutes.The suggestion structure that has shown different report things/signal ion among Fig. 2 c.Yet if for example in report thing part and/or balance portion, deuterium also is used for substituting one or more hydrogen atoms and/or usefulness ¹⁸O substitutes ¹⁶O, then can expect has more than 9 kinds of different compounds, thereby can carry out more than 9 heavy experiments.With reference to following Fig. 6 a and 6b, carried out more detailed discussion for this possibility that further substitutes of the analyte of the labelled reagent/mark of this general formula.

To at Fig. 2 a and those the similar same amount dystopy compounds shown in the 2b alternative group (R wherein ₁And R ₂Can be used for forming 6 yuan of rings) be illustrated among Fig. 3 a and the 3b.These labelled reagents can form the same group of 9 kinds of different report thing/signal ions, and its suggestion structure is shown among Fig. 2 c.Can easily prepare alternative structure and show that embodiment of the present invention has versatility with the required character of analyte analyzation.

Usually, some intermediates of the analyte of the analyte of labelled reagent, mark and described labelled reagent and/or mark can comprise its salt form and/or its hydrate forms representative by formula I compound:

Wherein Z can be-OH ,-SH ,-O ^-V ⁺,-S ^-V ⁺, can form the reaction active groups of the analyte of mark, the leaving group of reaction active groups with the functional group reactions of analyte, wherein said leaving group is left away during in the nucleophilicity functional group of described labelled reagent and analyte or with the analyte response of covalent bonding, wherein to atom or group V ⁺, X ₁, X ₂, R ₁, R ₂, Y, J, K and L be explained in more detail below.

Therefore, in some embodiments, the reaction of the labelled reagent that analyte can be by described analyte and formula I ' compound (comprising its salt form and/or its hydrate forms) representative and mark:

Wherein Z ' represents the reaction active groups of the analyte that can form mark with the functional group reactions of analyte or the leaving group of described reaction active groups, wherein said atom or radicals X ₁, X ₂, R ₁, R ₂, Y, J, K and L will be explained in more detail below.Described labelled reagent can use in groups, and wherein said group comprises isomerism and/or isobaric compound, and the analyte of mark can be isomerism and/or isobaric equally thus.

In addition, in some embodiments, so the analyte of mark can be by formula I " representative, comprise its salt form and/or its hydrate forms:

Wherein, Z " analyte of representative and described labelled reagent covalent bonding (may by other atom or the group of described reaction active groups), wherein said atom or radicals X ₁, X ₂, R ₁, R ₂, Y, J, K and L be explained in more detail below.

In addition, in some embodiments, the intermediate of described labelled reagent can be by formula I " ' representative, comprise its salt form and/or its hydrate forms:

Z wherein " ' representative-OH ,-SH ,-O ^-V ⁺Or-S ^-V ⁺, wherein said V ⁺, X ₁, X ₂, R ₁, R ₂, Y, J, K and L be explained in more detail below.Described intermediate can be used for for example producing labelled reagent disclosed herein, comprises that original position produces described labelled reagent, and the labelled reagent that wherein said original position produces can be used for labelled analyte.

In illustrated compound, group Y-J-represents to report the thing part.Therefore, isomerism and/or isobaric labelled reagent group can be encoded, make every kind of different labelled reagents of described group have identical gross mass but the group Y-J-of every kind of different labelled reagents of wherein said group is encoded especially, for example by utilizing one or more isotope enrichments position to carry out, thereby when the key between the remainder of the group J of group Y-J-and the analyte of described mark (or its fragment) ruptures, can form report thing ion (referring to Fig. 1 c) in mass spectrometer with unique qualities.The peak intensity of the ion relevant with report thing part (being signal ion or report thing ion) can be associated with the amount (being often expressed as concentration and/or amount) of the analyte of mark in the analyzed sample.Therefore, isomeric and/or isobaric labelled reagent group can be used for the analyte of two or more different samples of mark, wherein said labelled reagent can be because of different sample different and wherein said labelled reagent can comprise can with the related report thing part of sample of the analyte that produces described mark with unique qualities.Therefore, even the complex mixture of the analyte by mark that the marked product that mixes multiple different samples is obtained is analyzed, also the described analyte in the different samples with two or more of information of the existence of for example each report thing part and/or amount can be associated.

As described herein, in groups nine kinds or more isomerism and/or isobaric labelled reagent be can prepare, thereby 9 weights (9-plex) or more experiment (a kind of diagram of exemplary analysis is found among Figure 19 a and the 19b) allowed.For example, might identify simultaneously and/or quantitatively the analyte in 9 kinds of (or more kinds of) different samples that carry out separately mixing after the isolabeling not.Such analysis can realize by determine unique report thing ion (it can have the structure shown in Fig. 2 c) from the potpourri of 9 kinds of different samples (every kind of sample is by the different labelled reagent institute mark the group shown in Fig. 2 a/2b or the 3a/3b).Therefore, embodiment of the present invention are particularly suitable for the multiple analysis (multiplex) of complex sample compound.For example, embodiments more of the present invention can be used for Proteomic analysis and/or genome analysis, and are used for the correlation research relevant with genome analysis and Proteomic analysis.Embodiments more of the present invention also can be used for the micromolecule analysis, such as lipid, steroids or amino acid analysis.Can use the experimental analysis of isomeric and/or isobaric reagent to include but not limited to time course research, biomarker is analyzed, multiplexed protein matter group analysis, multidimensional protein identification techniques experiment (mudpit experiment), compatibility Pull-down tests (affinity pull-down), (referring to for example laid-open U.S. Patents application No.US 2005-0208550 A1) and multiple control experiment (multiple control experiments) are measured in posttranslational modification (PTM).

Description of drawings

It will be appreciated by those skilled in the art that following described accompanying drawing is only presented for purposes of illustration.Described accompanying drawing is not the scope that is intended to limit by any way the present invention's instruction.

Fig. 1 a is the ingredient of analyte and the illustrating of breaking property thereof of labelled reagent or mark.

Fig. 1 b is the general ingredient of analyte and the illustrating of breaking property thereof of exemplary labelled reagent or mark based on N methyl piperazine.

Fig. 1 c is identified the general ingredient of analyte and the illustrating of breaking property thereof of the mark of general formula.

Fig. 1 d is the explanation of the general formula of some exemplary isobaric labelled reagents.

Fig. 2 a is different from measuring illustrating of dystopy compound, and wherein said compound can form 9-reorganization (a 9-plex set) with compound shown in Fig. 2 b.

Fig. 2 b is different from measuring illustrating of dystopy compound, and wherein said compound can form the 9-reorganization with compound shown in Fig. 2 a.

Fig. 2 c is used to report illustrating that thing ion (being the signal ion) various may structures, and described report thing ion can be produced by the same amount dystopy compound shown in Fig. 2 a, 2b, 3a and the 3b.

Fig. 3 a is illustrating of the different dystopy of amount together compounds, and wherein said compound can form the 9-reorganization with compound shown in Fig. 3 b, and wherein said counterbalance comprises ring structure.

Fig. 3 b is illustrating of the different dystopy of amount together compounds, and wherein said compound can form the 9-reorganization with compound shown in Fig. 3 a, and wherein counterbalance comprises ring structure.

Fig. 4 a is the explanation of exemplary same amount dystopy compound.

Fig. 4 b is exemplary isomeric explanation with amount dystopy compound.

Fig. 5 is illustrating of process that the analyte of exemplary carrier-bound mark is disconnected from carrier.

Fig. 6 a is the illustrating of analyte of labelled reagent or mark, and wherein said report thing comprises a plurality of isotope enrichments position.

Fig. 6 b is the illustrating of analyte of labelled reagent or mark, and wherein said counterbalance (connector) comprises a plurality of isotope enrichments position.

Fig. 7 a is the explanation of the exemplary synthetic step 1-3 of exemplary indicia reagent.

Fig. 7 b is the explanation of the exemplary synthetic step 4-5 of exemplary indicia reagent.

Fig. 7 c is the explanation of the exemplary synthetic step 6-7 of exemplary indicia reagent.

Fig. 8 is the illustrating of scheme of synthetic various active ester.

Fig. 9 a is the table that illustrates isotope-coded raw material, and by putting into practice the present invention disclosed herein, described raw material can be used to prepare multiple with amount dystopy compound.

Fig. 9 b is the table that illustrates isotope-coded raw material, and by putting into practice the present invention disclosed herein, described raw material can be used to prepare multiple with amount dystopy compound.

Fig. 9 c is the table that illustrates isotope-coded raw material, and by putting into practice the present invention disclosed herein, described raw material can be used to prepare multiple with amount dystopy compound.

Figure 10 a is the table that illustrates isotope-coded raw material, and by putting into practice the present invention disclosed herein, described raw material can be used to prepare multiple with amount dystopy compound.

Figure 10 b is the table that illustrates isotope-coded raw material, and by putting into practice the present invention disclosed herein, described raw material can be used to prepare multiple with amount dystopy compound.

Figure 10 c is the table that illustrates isotope-coded raw material, and by putting into practice the present invention disclosed herein, described raw material can be used to prepare multiple with amount dystopy compound.

Figure 11 a be to the sarcosine derivative of coding a kind of may route of synthesis illustrate, the sarcosine derivative of described coding can be used for preparing the N-methyl-bridged piperazine derivatives of coding.

Figure 11 b be to the sarcosine derivative of coding a kind of may route of synthesis illustrate, the sarcosine derivative of described coding can be used for preparing the N-methyl-bridged piperazine derivatives of coding.

Figure 12 a is illustrating a kind of possibility route of synthesis of the N-methyl ethylenediamine of the coding that comprises two isotope-coded positions.

Figure 12 b is illustrating a kind of possibility route of synthesis of the N-methyl ethylenediamine of the coding that comprises four isotope-coded positions.

Figure 12 c is illustrating a kind of possibility route of synthesis of the N-methyl ethylenediamine of the coding that comprises four isotope-coded positions.

Figure 12 d is illustrating a kind of possibility route of synthesis of the piperazine of the coding that comprises four isotope-coded positions.

Figure 12 e is illustrating a kind of possibility route of synthesis of the piperazine of the coding that comprises two isotope-coded positions.

Figure 13 is illustrating a kind of possibility workflow of using isobaric labelled reagent group (being label 1 and label 2) processing specimen and control sample.

Figure 14 a is illustrating a kind of possibility workflow of using isobaric labelled reagent group (being label 1 and label 2) processing specimen and control sample.

Figure 14 b is illustrating the ingredient of catching holder.

Figure 14 c catches illustrating of holder to exemplary.

Figure 15 is illustrating a kind of possibility workflow of using isobaric labelled reagent group (being label 1 and label 2) processing specimen and control sample.

Figure 16 is illustrating a kind of possibility workflow of using two kinds of protein examples of isobaric labelled reagent group (being label 1, label 2, label 3 and label 4) processing.

Figure 17 is illustrating a kind of possibility workflow of using two kinds of protein examples of isobaric labelled reagent group (being label 1, label 2, label 3 and label 4) processing.

Figure 18 is illustrating a kind of possibility workflow of using two kinds of protein examples of isobaric labelled reagent group (being label 1, label 2, label 3, label 4, label 5 and label 6) processing.

Figure 19 a is to the label of MS analysis of the peptide of selected quality and illustrating of product (especially m/z), and the peptide of wherein said selected quality is derived from and adopts isobaric labelled reagent group to carry out not 9 kinds of different samples of isolabeling.

Figure 19 b is that the MS that wherein said peptide is selected from as shown in Figure 19 a analyzes to 9 kinds of the illustrating of MS/MS analysis result of the peptide of isolabeling (having specific m/z value) not.

Figure 20 is illustrating the possible approach of some exemplary indicia reagent.

Figure 21 is illustrating the possible approach of some exemplary indicia reagent.

Figure 22 a is to use the spectrogram of MS data of the peptide ([Glu1]-human fibrin peptide B) of the exemplary indicia reagent mark of described not coding form herein.

Figure 22 b is the MS/MS data spectrogram at the selected peak of the peptide of the mark shown in Figure 22 a, wherein described peptide is carried out the analysis again of CID fracture and fragment.

Figure 23 a is to use the MS data spectrogram of the peptide ([Glu1]-human fibrin peptide B) of the exemplary indicia reagent mark of described not coding form herein.

Figure 23 b is the MS/MS data spectrogram at the selected peak of the peptide of the mark shown in Figure 23 a, wherein described peptide is carried out the analysis again of CID fracture and fragment.

Figure 24 a is to use the MS data spectrogram of the peptide ([Glu1]-human fibrin peptide B) of the exemplary indicia reagent mark of described not coding form herein.

Figure 24 b is the MS/MS data spectrogram at the selected peak of the peptide of the mark shown in Figure 24 a, wherein described peptide is carried out the analysis again of CID fracture and fragment.

Figure 25 a is that wherein said reagent can form 8-reorganization (8-plex set) with compound shown in Figure 25 b to difference illustrating of amount dystopy reagent together.

Figure 25 b is that wherein said reagent can form the 8-reorganization with compound shown in Figure 25 a to difference illustrating of amount dystopy reagent together.

Figure 26 a is that wherein said reagent can form the 8-reorganization with compound shown in Figure 26 b to difference illustrating of amount dystopy reagent together.

Figure 26 b is that wherein said reagent can form the 8-reorganization with compound shown in Figure 26 a to difference illustrating of amount dystopy reagent together.

Figure 27 a is the explanation to the exemplary synthetic step 1-3 of exemplary indicia reagent.

Figure 27 b is the explanation to the exemplary synthetic step 4-5 of exemplary indicia reagent.

Figure 27 c is the explanation to the exemplary synthetic step 6-7 of exemplary indicia reagent.

Figure 28 a is illustrating a kind of possibility route of synthesis of the N-methyl ethylenediamine of the coding that comprises two isotope-coded positions.

Figure 28 b is to comprising six isotope-coded positions Fmoc-NH-CH ₂ CH ₂ -N (Me)- CO-CH ₂ CH ₂ -COOHSynthetic illustrating.

Figure 29 a is illustrating a kind of possibility route of synthesis of the N-methyl ethylenediamine of the coding that comprises four isotope-coded positions.

Figure 29 b is to comprising eight isotope-coded positions Fmoc-NH-CH ₂ CH ₂ -N (Me)- CO-CH ₂ CH ₂ -COOHSynthetic illustrating.

Whole documents and the similar material quoted in this application, include but not limited to patent, patented claim, article, books and internet page, no matter the form of these documents and similar material how, be any and all purposes, all clearly by reference and integral body is incorporated herein.

Definition

For explaining purpose of the present invention, will use to give a definition, if necessary, the term that whenever uses with odd number also comprises its plural form, and vice versa. For the purpose of explaining this specification and appended claims thereof, when the usage of the word in any definition proposed below and other any file (comprising any file that is incorporated herein by reference) is inconsistent, to always be as the criterion with the definition that proposes below, unless clearly illustrated that the opposite meaning (for example in the initial file that uses of term). Except as otherwise noted or " and/or " the obvious inappropriate situation of use under, the use of "or" herein always mean " and/or ". Except as otherwise noted or in the obvious inappropriate situation of the use of " one or more ", the use of " " herein always means " one or more ". " comprise ", the use of " comprising ", " containing " can exchange, and be not intended to limit. In addition, in the description to one or more embodiments, used term " to comprise " in the situation of (" comprising "), it will be appreciated by those skilled in the art that, under some concrete conditions, described one or more embodiments also can by phrase " basically by ... form " and/or " by ... form " be described.

A.) " analyte " used herein means measurable interested molecule. The non-limiting example of analyte includes but not limited to that protein, peptide, nucleic acid (DNA or RNA), carbohydrate, lipid, steroids and molecular weight are less than other little molecule of 1500 dalton (Da). The source of analyte or the sample that comprises described analyte do not limit it can be from any source. Analyte can be natural or synthetic. Analyte or the non-limiting example in source that comprises the sample of analyte comprise cell or tissue or its culture (or subculture). Other non-limiting example in analyte source includes but not limited to cell lysates thick or processing, body fluid, tissue extract, cell extract or from the separation process fraction (or part) of (separating or capillary electrophoresis separation such as chromatographic isolation, one dimension electrophoretic separation, two dimensional electrophoresis). Body fluid includes but not limited to the fluid of blood, urine, ight soil, brain liquid, amniotic fluid, lymph liquid or glandular secretion. The cytolysis thing of processing means except the needed processing of dissolved cell, also processes described cytolysis thing to carry out other processing to collected material. For example, described sample can be the cytolysis thing that comprises one or more analytes, and described analyte is by utilizing one or more proteolytic enzymes to process the peptide that the cytolysis thing forms with digestion precursor peptide and/or albumen.

B.) ester " mean ester and/or thioesters, unless clearly illustrate that really not so (for example when with reference to the concrete structure pointed out in addition) in its employed context.

C.) " fracture " used herein mean the fracture of covalent bond.

D.) " fragment " used herein/" fracture " (" the fragment ") product (noun) that means to rupture or the operation (verb) that causes fracture.

E.) " hydrate forms " used herein mean any hydration status of compound or mixture or compound more than a kind of hydration status. For example, described labelled reagent can be semihydrate, monohydrate, dihydrate etc. herein. In addition, the sample of described labelled reagent can comprise the form of monohydrate, dihydrate and semihydrate simultaneously herein.

F.) halogen group used herein refer to-F ,-Cl ,-Br or-I.

G.) herein to compound employed " isotope enrichment " mean enrichment one or more heavy atom isotopes (stable isotope for example, such as deuterium,¹³C、 ¹⁵N、 ¹⁸O、 ³⁷Cl or⁸¹Br) compound (for example labelled reagent). In some embodiments, also (for example can use unsettled isotope¹⁴C or³H). The amount that " enrichment " means heavy atom isotope has surpassed the abundance of natural isotope. In different embodiments, the compound of isotope enrichment can comprise the position of 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30 or more isotope enrichments.

Because isotope enrichment be not 100% effectively, be in less enrichment state and have low-qualityer impurity so in the sample of compound, can exist. Equally, because can there be the impurity of larger quality in too enrichment (enrichment of for example not expecting) and because the abundance of natural isotope in the sample of compound. In some embodiments, every kind of heavy atom isotope introducing can at least 80% isotopic purity be present in the isotope enrichment position. In some embodiments, every kind of heavy atom isotope introducing can at least 93% isotopic purity be present in the isotope enrichment position. In some embodiments, every kind of heavy atom isotope introducing can at least 96% isotopic purity be present in the isotope enrichment position. In some embodiments, every kind of heavy atom isotope introducing can at least 98% isotopic purity be present in the isotope enrichment position.

H.) " isotope enrichment position " used herein means the position in the compound, uses the light isotope of alternative this atom of heavy atom isotope at described position (for example¹³C substitutes¹²C， ¹⁸O substitutes¹⁶O， ¹⁵N substitutes¹⁴N, perhaps deuterium instead of hydrogen).

I.) employed " gently " means not carry out with heavy atom isotope the compound of enrichment for compound herein. Employed " gently " means the isotope of described atom minimum quality for atom herein. Mean to carry out with at least a heavy atom the compound of isotope enrichment herein for compound employed " weight ". Mean the heavy Indium isotopes of described atom herein for atom employed " weight ".

J.) " labelled reagent " used herein means to be suitable for the structure division that mark is used for the analyte of mensuration. Terms tag thing (" label ") and term tag (" tag ") and mark (" mark ") and other term of equal value and phrase are synonyms. For example, the analyte of mark (labeled analyte) can also be called the analyte (tagged analyte) of mark or the analyte (marked analyte) of mark. Therefore, the derivative of term " label " (" label "), " label " (" tag "), quality tab (" mass tag "), mark (" mark ") and these terms is of equal value with interchangeable, and refer to be suitable for mark or mark be used for the structure division of the analyte measured. Sometimes labelled reagent can be described as tagging reagents (" tagging reagent ") or mass tagging reagents (" mass-tagging reagent ").

K.) " natural isotopic abundance " used herein means, based on natural dominant one or more isotopes of occurring in nature, and the level of one or more heavy isotopes of in compound, finding (or distribution). For example, the native compound that derives from the living plant material usually comprise with respect to¹²About 1.08% of C¹³C。

L.) used herein is the structurally undistinguishable compound (except the isotopic content and/or distribution of heavy atom isotope) with identical nominal gross mass with amount dystopy thing (isobar). Be avoidance of doubt, according to definition herein, the compound 1-4 of Fig. 4 a is isobaric. Comparatively speaking, isomers used herein is the compound that structurally can distinguish with identical nominal gross mass. The same amount dystopy thing of exemplary isomery illustrates in Fig. 4 b.

M.) " carrier " used herein, " solid phase carrier " (" solid support ", " solid carrier ") or resin mean any solid phase material. Solid phase carrier comprises that term is such as " carrier " (" support "), " synthetic vectors ", " solid phase ", " surface ", " film " and/or " carrier " (" support "). Solid phase carrier can be comprised of organic polymer, such as polystyrene, polyethylene, polypropylene, polyvinyl fluoride, polyoxyethylene and polyacrylamide, with and copolymer and graft. Solid phase carrier can also be inorganic matter, such as glass, silica, controlled pore glass (CPG) or anti-phase silica. The profile of solid phase carrier can be the form on pearl, ball, particulate, particle, gel, film or surface. The surface can be the plane plane, basic or nonplanar. Solid phase carrier can be porous or non-porous, and can have expansion or unexpansive character. Solid phase carrier can be designed to the form of pond, depression or other container, pipe, feature or position. A plurality of solid phase carriers can be set to be in the diverse location of array, are addressable to automatically sending of reagent, or by detection method and/or equipment addressing.

N.) " sample or its part " used herein or " sample part " can be used for referring to the part of sample. The part of sample can obtain by the part of simple taking-up sample, and perhaps it can obtain by the separation process that makes sample be classified to two or more parts. Unless point out in addition in the content of specification, these terms are equivalence and interchangeable, mean the part of any type of sample generation.

O.) " signal ion " used herein and " report thing ion " can exchange, and all refers to by the analyte fracture that makes labelled reagent or mark by the report thing ion with unique qualities of reporting that thing partly produces. Signal ion or report thing ion are identified the labelled reagent of described uniqueness for labelled analyte, its peak intensity in MS/MS analyzes can be present in analytic sample in the amount of analyte of mark relevant. Signal ion used herein or report thing ion sometimes only are called the report thing. Report thing part used herein sometimes also only is called the report thing. Be to be understood that, the report thing partly refers to the group that combines with analyte or its fragment of labelled reagent, mark, and report thing ion refers to connect the fragment that the key of the analyte of described report thing part and described labelled reagent, mark or its fragment forms by fracture. To show its literal sense when therefore, using " report thing " in this article. It is also understood that phrase " unique report thing part " and " the report thing part with unique qualities " are equivalence and interchangeable, " unique report thing ion " is the also interchangeable of equivalence with " the report thing ion with unique qualities ".

P.) term used herein " salt form " comprises the salt of compound or the salt mixture of compound. In addition, the zwitterionic form of compound is also contained in the term " salt form ". Has the salt of compound of amine or other basic group such as obtaining with the reaction of suitable organic acid or inorganic acid (such as hydrochloric acid, hydrobromic acid, acetic acid, perchloric acid etc.) by making it. Compound with quaternary ammonium group also can contain counter ion such as chlorion, bromide ion, iodide ion, acetate ion, perchlorate etc. Salt with compound of carboxylic acid or other acidic functionality can make by making the reaction of described compound and suitable alkali (for example hydroxide bases). Therefore, the salt of acid functional group can have counter ion, such as sodium ion, potassium ion, magnesium ion, calcium ion etc.

Q.) " synthetic compound " used herein means to comprise the compound that the natural development ways of manipulation produces by manipulation process. Therefore, can utilize synthesising chemical technology to prepare synthetic compound. Yet, " synthetic compound " used herein also means to comprise the compound that for example produces by enzyme method, described enzyme method comprises the compound that isotope enrichment for example is provided to organism (such as bacterium or yeast), produces the labelled reagent of described isotope enrichment herein or the intermediate of described labelled reagent thereby described organism changes these compounds.

R.) " synthetic enrichment " used herein refer to handle synthetic or thus natural process produces the labelled reagent of described isotope enrichment herein or the intermediate of described mark preparation.

S.) generally acknowledged, the quality of atom or molecule can be similar to, and usually is immediate whole atomic mass unit or immediate in ten or with hundred atomic mass unit." gross mass " used herein refers to absolute mass and the Approximation Quality in following scope, to such an extent as to the isotopic use of different atomic types obtains and no matter can employed different isotope type detect in very little qualitatively difference being of equal value near them on function on the purpose of the quality of described report thing of balance and/or connector part (thereby the gross mass of described report thing/connector combination is identical in the group of isomerism and/or isobaric labelled reagent or kit) so qualitatively in the described scope.

For example, the common isotope of oxygen has the gross mass of 16.0 (actual mass 15.9949) and 18.0 (actual mass 17.9992), the common isotope of carbon has the gross mass of 12.0 (actual mass 12.00000) and 13.0 (actual mass 13.00336), and the common isotope of nitrogen has the gross mass of 14.0 (actual mass 14.0031) and 15.0 (actual mass 15.0001).Though these values are estimated, it will be understood to those of skill in the art that if use at the isotope enrichment position in the label in group ¹⁸The O isotope, so extra 2 mass units (oxygen isotope that exceeds gross mass 16.0) can for example comprise described by the label of introducing other ¹⁶Compensate in the different labels of O group: two carbon ¹³The C atom substitutes two ¹²The C atom, two ¹⁵The N atom substitutes ¹⁴N, even one ¹³C atom and one ¹⁵The N atom substitutes ¹²C and ¹⁴N gives with compensation ¹⁸O.By the method, described group of two interior different labels can have identical gross mass, because using two ¹³The C atom (substitutes two ¹²The C atom), two ¹⁵The N atom (substitutes two ¹⁴The N atom), one ¹³C and one ¹⁵N (substitutes ¹²C and ¹⁴N) or one ¹⁸The O atom (substitutes one ¹⁶The O atom) thus make all labels of described group or kit when increasing by two dalton qualitatively, in fact the difference in quality between them very little, can not hinder described analysis in fact.

This point can obtain explanation with reference to figure 4a.In Fig. 4 a, compound 3 (Fig. 4 a; Molecular formula C ₅ ¹³CH ₁₁ ¹⁵N ₂O) report thing/connector combination has two ¹⁵N atom and one ¹³The C atom, premier's opinion quality is 130.085.Comparatively speaking, with amount dystopy thing 1 (Fig. 4 a; Molecular formula C ₅ ¹³CH ₁₁N ₂ ¹⁸O) has one ¹⁸O atom and one ¹³The C atom, premier's opinion quality is 130.095.Although partly there is small absolute mass difference (quality 130.095 with quality 130.085) in described report thing/connector, compound 1 and 3 can be with amount dystopy thing, they except the content of heavy atom isotope, undistinguishable structurally.Yet, for purposes of the present invention, the gross mass of compound 1 and 3 report thing/connector part all is 130.1, because no matter whether mass spectrometer is sensitive in being enough to measure with the fine difference between the absolute mass of measuring the report thing ion that dystopy thing 1 and 3 produced, can not counteract analysis.

Equally, with reference to figure 4b, for example understand two kinds isomeric with amount dystopy things, wherein the quality of compound 5 and report thing/connector part of 6 is respectively 144.111 and 144.100.For purposes of the present invention, the gross mass of the report thing of these compounds/connector part all is 144.1, because no matter whether mass spectrometer is sensitive in being enough to measure with the little difference between the absolute mass of measuring the report thing ion that dystopy thing 5 and 6 produced, can not counteract analysis.

Clearly visible from Fig. 4 a and 4b, identical heavy atom isotope is not unique Consideration in the distribution in the structure for the group that produces isomerism and/or isobaric labelled reagent.Can be with isomers and/or the same amount dystopy thing of heavy atom isotope type hybrid to realize desired gross mass.Like this, the selection of heavy atom isotope (combination) and to distribute all be admissible factor when preparation can be used for the isomerism of embodiment of the present invention and/or isobaric labelled reagent.

T.) term used herein " alkyl " refers to the C of straight or branched ₂-C ₈Hydrocarbon or ring-type C ₃-C ₈Hydrocarbon (being naphthenic base, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cyclohexylmethylene), it can be fully saturated.When using in this article, term " alkyl " refers to be substituted or unsubstituted group.Term " alkyl " also mean on the alkyl chain wherein one or more methylene can by heteroatoms as-O-,-Si-or-those compounds that S-replaces.In some embodiments, alkyl can be the C of straight or branched ₂-C ₆Hydrocarbon or ring-type C ₃-C ₆Hydrocarbon, it can be fully saturated.

U.) term used herein " alkylidene " refers to comprise the alkyl chain of straight or branched of at least two points that are connected at least two structure divisions or group of naphthene base ({ CH for example ₂}-(methylene) ,-{ CH ₂CH ₂}-(ethylidene),

Deng, the point that its bracket { } expression connects).When using in this article, term " alkylidene " refers to be substituted or unsubstituted group.Term " alkylidene " also mean on the alkyl chain of alkylidene wherein one or more methylene (if any) can by heteroatoms as-O-,-Si-or-those compounds that S-replaces.In some embodiments, alkylidene can be C ₁-C ₁₀Hydrocarbon.In some embodiments, alkylidene can be C ₂-C ₆Hydrocarbon.

V.) term used herein " thiazolinyl " refers to comprise the straight or branched C of one or more pairs of keys ₂-C ₈Hydrocarbon or ring-type C ₃-C ₈Hydrocarbon.When using in this article, term " thiazolinyl " refers to be substituted or unsubstituted group.Term " thiazolinyl " also mean on the alkyl chain of thiazolinyl wherein one or more methylene (if any) can by heteroatoms as-O-,-Si-or-those compounds that S-replaces.In some embodiments, thiazolinyl can be the C with straight or branched of one or more pairs of keys ₂-C ₆The C of hydrocarbon or ring-type ₃-C ₆Hydrocarbon.

W.) term used herein " alkenylene " refers to comprise the thiazolinyl that is connected to two points at least two parts.When using in this article, term " alkenylene " refers to be substituted or unsubstituted group.Term " alkenylene " also mean on the alkyl chain of alkenylene wherein one or more methylene (if any) can by heteroatoms as-O-,-Si-or-those compounds that S-replaces.

X.) term used herein " alkynyl " refers to comprise the straight or branched C of one or more three keys ₂-C ₈Hydrocarbon or ring-type C ₃-C ₈Hydrocarbon.When using in this article, term " alkynyl " refers to be substituted or unsubstituted group.Term " alkynyl " also mean on the alkyl chain of alkynyl wherein one or more methylene (if any) can by heteroatoms as-O-,-Si-or-those compounds that S-replaces.In some embodiments, alkynyl can be the C with straight or branched of one or more three keys ₂-C ₆The C of hydrocarbon or ring-type ₃-C ₆Hydrocarbon.

Y.) term used herein " alkynylene " refers to comprise the alkynyl that is connected to two points at least two parts.When using in this article, term " alkynylene " refers to be substituted or unsubstituted group.Term " alkynylene " also mean on the alkyl chain of alkynylene wherein one or more methylene (if any) can by heteroatoms as-O-,-Si-or-those compounds that S-replaces.

Z.) term used herein " aliphatics " refers to alkyl, thiazolinyl and the alkynyl part of any straight chain as defined above, side chain or ring-type.When using in this article, term " aliphatics " refers to be substituted or unsubstituted group.

Aa.) term used herein " aryl " (separately or as another structure (for example aryl alkyl etc.) a part of time) refers to carbocyclic ring aromaticity group, such as phenyl.Aryl also comprises the fragrant ring system of the many cyclophanes that condense, wherein carbocyclic ring aromatic rings and other carbocyclic ring aromatic rings condense (for example 1-naphthyl, 2-naphthyl, 1-anthryl, 2-anthryl etc.), and perhaps wherein carbocyclic ring aromatic rings and one or more carbocyclic ring non-aromatic ring condense (for example naphthane, indane etc.).Term used herein " aryl " refers to be substituted or unsubstituted group.

Ab.) term used herein " heteroaryl " refers to comprise 1,2,3 or 4 heteroatomic aromatic heterocycle that is independently from each other nitrogen, sulphur and oxygen.Term used herein " heteroaryl " refers to be substituted or unsubstituted group.Heteroaryl can condense with one or two ring, such as naphthenic base, aryl or hetero-aromatic ring.The tie point of heteroaryl and molecule can be heteroaryl, naphthenic base, Heterocyclylalkyl or aryl rings, and described heteroaryl can connect by carbon atom or heteroatoms.The example of heteroaryl comprises imidazole radicals, furyl, pyrrole radicals, thienyl, thiazolyl isoxazolyl, isothiazolyl, thiadiazolyl group oxadiazole base, pyridine radicals, pyrimidine radicals, pyrazinyl, pyridazinyl, quinolyl, isoquinolyl, indazolyl benzoxazolyl, the benzoisoxazole base, benzofuranyl, benzothiazolyl, the indolizine base, imidazopyridyl, pyrazolyl, triazolyl oxazolyl, tetrazole radical, benzimidazolyl, the benzisothiazole base, diazosulfide base Ben Bing oxadiazole base, indyl, the tetrahydro indole base, azaindolyl (azaindolyl), imidazopyridyl, quinazolyl, purine radicals, pyrrolo-[2,3] pyrimidine radicals, pyrazolo [3,4] pyrimidine radicals or benzo (b) thienyl, wherein each group all can randomly be substituted.

Ac.) term used herein " arlydene " refers to comprise aryl or the heteroaryl (for example phenylene etc.) that is connected at least two points on two structure divisions at least.The tie point that arlydene and carbocyclic ring non-aromatic ring condense can be in aromatic rings, non-aromatic ring any on.Term used herein " arlydene " refers to be substituted or unsubstituted group.

Ad.) term used herein " aryl alkyl " refers to the aryl or the heteroaryl that are connected with other structure division by the alkylidene connector.Term used herein " aryl alkyl " refers to be substituted or unsubstituted group.Term " aryl alkyl " mean on the alkyl chain of aryl alkyl wherein one or more methylene (if any) can by heteroatoms as-O-,-Si-or-those compounds that S-replaces.

Ae.) term used herein " arlydene alkyl " refers to have the aryl alkyl of at least two points that link to each other with at least two structure divisions.Connect second can be in aromatic ring or alkylidene any on.Term used herein " arlydene alkyl " refers to be substituted or unsubstituted group.Term " arlydene alkyl " also mean on the alkyl chain of arlydene alkyl wherein one or more methylene (if any) can by heteroatoms as-O-,-Si-or-those compounds that S-replaces.When the arlydene alkyl was substituted, substituting group can be on the aromatic rings of described arlydene alkyl or any or two in the alkylene moiety.

Af.) term used herein " optional replacement " and " replacement or unsubstituted " are of equal value and interchangeable.The suitable substituent of any alkyl, alkylidene, thiazolinyl, alkenylene, alkynyl, alkynylene, aryl, aryl alkyl, arlydene, heteroaryl or arlydene alkyl is included in the embodiment of the present invention any substituting group stable under the employed reaction conditions.The non-limiting example of suitable substituent can comprise alkyl (for example methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, isobutyl, sec-butyl, the tert-butyl group, cyclohexyl etc.), haloalkyl (trifluoromethyl, 2 for example, 2,2-trifluoroethyl etc.), alkoxy (for example methoxyl, ethoxy etc.), aryl (for example phenyl), aryl alkyl (for example benzyl), nitro, cyano group, quaternised nitrogen-atoms or halogen group (for example fluorine, chlorine, bromine and/or iodine).

In addition, any part of alkyl, alkylidene, thiazolinyl, alkenylene, alkynyl, alkynylene, aryl, aryl alkyl, arlydene, heteroaryl or arlydene alkyl also can by=O or=S replaces.

Ah.) term used herein " active ester " refers to be easy to obtain respectively with amine, pure and mild some thiol reactant the compound of acid amides, ester and thioesters under alkali condition.Please refer in addition: Leo A Paquette, Encyclopedia of Reagents for Organic Synthesis.Vol.2, John Wiley and Sons, New York, 1995, it shows that as evidence active ester is a term used for a long time in the organic chemistry filed.

Ai.) term used herein " heterocycle " refers to comprise any ring shaped molecule structure of at least two kinds of different atoms of elements in described one or more rings.Please refer in addition: OxfordDictionary of Biochemistry and Molecular Biology, Oxford UniversityPress, Oxford, 1997, it shows that as evidence heterocycle is a term used for a long time in the organic chemistry filed.

Aj.) term used herein " leaving group " refers to any electrically charged or uncharged atom or the group of leaving away in substitution reaction or displacement reaction, and it is considered to the residue or the major part of response matrix.Please refer in addition: Oxford Dictionary of Biochemistry andMolecular Biology, Oxford University Press, Oxford, 1997, it shows that as evidence leaving group is a term used for a long time in the organic chemistry filed.

Ak.) term used herein " blocking group " refer to molecule in functional group reactions and combine with it preventing that described functional group is subjected to the chemical group of the influence of the reaction that this molecule participates in subsequently, thereby it can be removed the unprotected functional group of regenerating subsequently.Please refer in addition: OxfordDictionary of Biochemistry and Molecular Biology, Oxford UniversityPress, Oxford, 1997, it shows that as evidence blocking group is a term used for a long time in the organic chemistry filed.

Specific embodiments

Should be appreciated that following discussion at this " summary " part can relate to some or all of various embodiments of the present invention.

1. summary

Labelled reagent:

As previously mentioned, labelled reagent comprises report thing part, counterbalance part (or connector part) and reaction active groups usually (Fig. 1 a).Novel markings reagent more disclosed herein can be by general formula I ' representative, comprise its salt form and/or its hydrate forms:

Report thing counterbalance

Wherein Z ' represents the leaving group of reaction active groups or described reaction active groups, wherein said atom or radicals X ₁, X ₂, R ₁, R ₂, Y, J, K and L be explained in more detail below.For the compound of formula I ' representative, marked the report thing part and the counterbalance part of described molecule.The formula of other isobaric labelled reagent is found among Fig. 1 d.

Reaction active groups:

The reaction active groups of the labelled reagent that uses in the embodiment of method, potpourri, kit and/or composition (sometimes with abbreviation " RG " expression) can be the group of electrophilic or nucleophilicity, its can with one or more functional group reactionses of one or more reactivity analytes of sample.Should be appreciated that in some cases, can think that reaction active groups comprises atom or the group that combines with connector (counterbalance).For example; if reaction active groups is active ester or acyl halide group; so for the purpose of the quality of the report thing in balance isomerism and/or isobaric labelled reagent group part; the carbonyl of described active ester or acyl halide also can be considered to combine with connector in some cases, and wherein carbonyl carbon is present in the analyte of labelled reagent and mark.Therefore, in some embodiments, reaction active groups can be regarded as the leaving group of only representing reaction active groups.

It should also be understood that, when reaction active groups is partly represented by more following concrete structures, analyte can be connected with connector (counterbalance) by one or more other atom or groups, described other a atom or the group part that can be considered to or not be considered to described connector (counterbalance).

Reaction active groups can be pre-existing in or it can prepare in position.The in-situ preparing of reaction active groups can be carried out under the situation that lacks the reactivity analyte or it can carry out under the situation that has the reactivity analyte.For example, hydroxy-acid group can be with water miscible carbodiimide (1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride for example; EDC) thus carrying out in-situ modification makes the electrophilicity group, described electrophilicity group can react such as alkyl or aryl amine with the nucleophilic thing of analyte.In some embodiments, utilize EDC to the activation of the hydroxy-acid group of labelled reagent can contain amine (nucleophilic thing) analyte in the presence of carry out.In some embodiments, described amine (nucleophilic thing) analyte that contains adds after also can carrying out at the initial reaction with EDC.In other embodiments, can remove blocking group and original position generation reaction active groups by original position.Therefore, can reckon with any existence or the new reagent that produces of impact analysis thing derivatization by the reaction of nucleophilic thing and/or close electric thing by method, potpourri, kit and/or the composition of embodiment of the present invention.

Reaction active groups at labelled reagent is under the situation of close electric thing, and the electric thing of described parent can react with the suitable nucleophilic group of one or more analytes.Reaction active groups at labelled reagent is under the situation of nucleophilic thing, and described nucleophilic thing can react with the suitable electrophilic group of one or more analytes.Many suitable nucleophilic groups and electrophilic group to be the chemistry and biochemical field in known and commonly used.Comprise that the non-limiting example with the reagent of the suitable nucleophilic of realizing its derivatization or electrophilic group that can combine with analyte (for example protein, peptide, nucleic acid, carbohydrates, lipid, steroids or quality are less than 1500 daltonian other micromolecule) is described in PierceLife Science ﹠amp; Analytical Research Products Catalog ﹠amp; Handbook (aPerstorp Biotec Company), Rockford, IL 61105, among the USA.Other suitable reagent is well known in the art, and they can be commercially available by a plurality of other suppliers such as Sigma-Aldrich place.

The reaction active groups of labelled reagent can be the amine reaction active groups.For example, the amine reaction active groups can be an active ester.Active ester is well-known in peptide is synthetic, and it refers at some ester that is generally used for can being easy to react with amino acid whose N-α amine under the synthetic condition of peptide (referring to Leo A Paquette, Encyclopedia of Reagents for Organic Synthesis, Vol.2.John Wiley and Sons, New York, 1995).Described amine active ester group can be N-hydroxy-succinamide ester (NHS), N-hydroxysulphosuccinimide ester (NHSS), pentafluorophenyl group ester (Pfp), 2-nitrobenzophenone ester, 3-nitrobenzophenone ester (3-NP), 4-nitrobenzophenone ester (4-NP), 2,4-dinitrophenyl ester, pentafluorophenyl group ester (Pfp), five chlorophenyl ester (Pcp), 3-hydroxyl-1,2,3-phentriazine-4 (3H)-ketone ester (Dhbt), hydroxyl pyrrolidine ketone ester (NHP), 2,4-dihalogenated phenyl ester is (referring to Fig. 8 with lower banner: the content under " the labelled reagent preparation method illustrates "), 2,2,2-trifluoroethyl ester or 1,1,1,3,3,3-hexafluoro-2-propyl diester.For example, the leaving group of active ester (be commonly referred to Z ' in some embodiments of this paper, such as in this case, variable R G partly is a synonym with the leaving group of reaction active groups only) can be by following various representative:

Wherein X ' be-O-or-S-, and each X " be independently of one another-F ,-Cl ,-Br or-I (the synthetic more detailed description about the active ester of representative compounds sees also U.S. publication application No.US 2005-0148771 A1).Foregoing description all be the alcohol or the mercaptan leaving group of active ester, the carbonyl carbon reaction that wherein said alcohol or mercaptan leaving group can be by amino acid whose N-α-amine and described active ester groups and being replaced.Those of ordinary skills should be understood that, can utilize known method and herein disclosure prepare any suitable mark/tagging reagents described herein active ester (for example N-hydroxy-succinamide base ester) (for example please refer in addition: GregT.Hermanson (1996). ＂ The Chemistry of Reactive Groups ＂ in ＂ Bioconjugate Techniques ＂ Chapter 2 pages 137-165, Academic Press, (New York); Also please refer to: Innovation And Perspectives In Solid PhaseSynthesis, Editor:Roger Epton, SPCC (UK) Ltd, Birmingham, 1990).

In some embodiments, the reaction active groups of labelled reagent can be a mixed acid anhydride, thereby produces amido link because known mixed acid anhydride and amine groups are reacted effectively.Mixed acid anhydride be known and can utilize organic chemistry and/or the chemistry of peptides field in method commonly used be prepared.

In some embodiments, the reaction active groups of labelled reagent can be an acyl halide group, such as acyl fluorides group (referring to J.Am.Chem.Soc. such as Carpino, 112:9651 (1990)) or acid chloride groups.

In some embodiments, the reaction active groups of labelled reagent can be the thiol reactant reactive group.For example, described thiol reactant reactive group can be aryl halide, alpha-halogen thioketones or the alpha-halogen imines of maleimide, alkyl halide, alpha-halogen acyl group.Halogenide or halo mean the atom of fluorine, chlorine, bromine or iodine.Described thiol reactant reactive group is well-known and can utilizes method commonly used in the chemistry of peptides field to be prepared.

In some embodiments, the reaction active groups of labelled reagent can be the hydroxyl reaction reactive group.For example, described hydroxyl reaction reactive group can be trityl halogenide or silyl halides active part.Described trityl halogenide active part can be replace (for example X " '-methoxyl trityl, X " '-dimethoxytrityl, X " '-trimethoxy trityl etc.) or unsubstituted, wherein X is the key of coupled reaction reactive group and connector (being counterbalance).Described silicyl active part can be the silyl halides that alkyl replaces, such as X " '-dimetylsilyl, X " '-two triethylsilyl (X " '-ditriethylsilyl), X " '-dipropyl silicyl, X " '-diisopropyl silicyl etc.), X wherein " ' be the key of coupled reaction reactive group and connector (being counterbalance).Described reaction active groups is well-known and can utilizes method commonly used in the nucleic acid chemistry field to be prepared.

In some embodiments, the reaction active groups of labelled reagent can be the nucleophilic thing, such as amino, hydroxyl or mercapto.In some embodiments, described necleophilic reaction reactive group can be aminoalkyl, hydroxy alkyl or alkylthio.Described reaction active groups is well-known and can utilizes method commonly used in the organic chemistry filed to be prepared.

Report thing part:

The report thing part of the labelled reagent that uses in the embodiment of the present invention (sometimes using abbreviation " RP " expression) can be the group with the unique qualities (or mass-to-charge ratio) that can measure.Therefore, every group report thing part can have unique gross mass.Different report thing parts can comprise one or more heavy atom isotopes to reach its unique gross mass.For example, exist carbon ( ¹²C, ¹³C and ¹⁴C), nitrogen ( ¹⁴N and ¹⁵N), oxygen ( ¹⁶O and ¹⁸And can be used for preparing not on the same group report thing part O) or the isotope of hydrogen (hydrogen, deuterium and tritium).These are not to be restrictive, because other light atom and heavy atom isotope also can be used for reporting the thing part.The basic initiation material that is suitable for preparing the report thing part that comprises light and heavy atom isotope can be buied from each commercial source, such as Cambridge Isotope Laboratories, Andover, MA (referring to list or " the basic initiation material " of www.isotope.com) and Isotec (branch offices of Sigma-Aldrich).Cambridge Isotope Laboratories and Isotec also can prepare needed compound according to the synthetic agreement of client.The same.

Report thing part can comprise fixing electric charge or can be ionized.Because report thing part can comprise fixing electric charge or can be ionized, so labelled reagent is may be with salt (or potpourri of salt) or zwitterionic form separated or be used for labeled reactant activity analysis thing.The ionization of report thing part (or report thing ion) can be measured it in mass spectrometer.Therefore, the existence of report thing part in the analyte of mark can be used as fragment ions (being sometimes referred to as signal ion (or report thing ion)) and measures.After ionization, report thing ion can comprise one or more clean positive charges or net negative charge.Therefore, report thing ion can comprise one or more acidic-groups and/or basic group, because these groups can ionization easily in mass spectrometer.For example, report thing part can comprise one or more nitrogen-atoms (positive charge) or one or more ionizable acid group, such as hydroxy-acid group, sulfonic acid group or phosphate group (negative charge).The non-limiting example that comprises the report thing part of at least a basic nitrogen comprises and replacing or the unsubstituted compound that contains morpholine, piperidines or piperazine.

Unique report thing part can combine with interested sample, thereby label has one or more analytes of the sample of described unique report thing part.Like this, can be associated with the information of a kind of or whole analytes of relevant sample about the information of unique report thing part (being detected as report thing ion usually).Yet, when measuring report thing ion, the report thing part of described uniqueness needn't be connected with the analyte physical property, thereby but the ion of the analyte of mark ruptured produce sub-fragment ions (daughter fragment ion) and report thing ion after, the gross mass of the uniqueness of report thing ion for example can be measured in the quality analysis second time of the mass-synchrometer of series connection.The report thing ion of measuring can be used for identifying the sample that the analyte of mensuration is originated.In addition, the report thing ion of described uniqueness is with respect to other relative quantity and/or absolute magnitude (often representing with concentration and/or amount) of reporting thing ion or can be used for analyte in the working sample with respect to the amount of the report thing ion relevant with calibration standard (for example with the concrete analyte of reporting substance markers).Therefore, such as the information of the amount of one or more analytes in the concrete sample can with the report thing part correlation connection that is used for every kind of concrete sample of mark.Under the situation also determined to the evaluation of one or more analytes, described information can be associated with the information that relates to different report thing ions, thereby helps the evaluation of the analyte of every kind of mark in one or more samples and the mensuration of amount.

Report thing part can comprise the nitrogen-atoms with the mesomethylene carbon covalent bonding of replacement or the alkylating acetate part of unsubstituted N-, wherein said replacement or unsubstituted methylene group but not hydroxy-acid group is the part of report thing.Hydroxy-acid group can be used for report thing and connector are coupled together.Nitrogen-atoms can be by one, two or three group alkylations.For example, the part that contains nitrogen-atoms can be the secondary amine that replaces, such as dimethylamine, diethylamine or di-n-propylamine.

Report thing part can be 5,6 or 7 yuan of heterocycles that comprise theheterocyclic nitrogen atom, described theheterocyclic nitrogen atom is substituted or unsubstituted acetate part N-alkylation, analyte is connected on the acetate part by the carbonyl carbon of N-acetate alkyl part, wherein said replacement or unsubstituted methylene group but not hydroxy-acid group is the part of report thing.Heterocycle can be fragrance or non-fragrance.Therefore, report thing part can be represented that wherein group Y represents 5,6 or 7 yuan of heterocycles by formula Y-J-, and group J represents the replacement or the unsubstituted methylene of acetate part.Heterocycle can be that replace or unsubstituted.For example, the substituting group of heterocyclic moiety can comprise alkyl, alkoxy and/or aryl.Substituting group can comprise protection or unprotected group, such as being suitable for analyte is connected to amine, hydroxyl or thiol group on the carrier.Heterocycle can comprise other heteroatoms such as one or more silicon, nitrogen, oxygen and/or sulphur atom.

Can select to report that the thing part is not so that inferior fracture (sub-fragment) takes place basically for it under the usual conditions that analyte is analyzed.For clarity sake, this is an optional and nonessential feature.Can select to report thing, make analyte at mark in mass spectrometer apply that inferior fracture does not take place the report thing basically under the condition that dissociation energy makes its fracture." inferior fracture not taking place basically " and mean when interested analyte is carried out successful analysis, is difficult to maybe can not detect the fragment of report thing under the above-mentioned background noise.

In some embodiments, can have a mind to select, make the gross mass of report thing ion be different from the quality of any expectation fragment of the quality of the analyte of seeking to measure or analyte.For example, be under the situation of analyte at protein or peptide, can select to report the gross mass of thing ion, make it to be different from any naturally occurring amino acid or peptide, or its fragment ions of estimating.This can help the mensuration of analyte, because based on analyte, the shortage with any possibility sample composition of same consistent quality can increase the confidence level of any analysis result.The example of the mass range of peptide is found in the table 1, and wherein seldom background is measurable arrives.

Table 1: possible " No Tooting Area " of selecting the label fragment ions m/z relevant with peptide analysis

M/z begins-finishes
10-14
	19-22
24-26
	31-38
40-40
	46-50
52-52
	58-58
61-69
	71-71
74-83
	89-97
103-109
	113-119
121-125
	128-128
131-135
	137-147
149-154
	156-156
160-174
	177-182
184-184
	188-189
191-191
	202-207
210-210
	216-222
224-226

Report thing part can be non-polymeric.Can select to report that the thing part is to produce the signal ion of m/z less than 250 atomic mass units (amu).Can select to report that the thing part is to produce the signal ion of m/z less than 200amu.Can select to report that the thing part is to produce the signal ion of m/z less than 150amu.These micromolecule can easily be measured in the mass spectrophotometry of the second level, and it does not contain other the sample composition that has same consistent quality in the one-level mass spectrophotometry.In this article, can carry out the second order ms analysis, in tandem mass spectrometer (perhaps, for example by the post-source decay in the single-stage instrument), carry out usually the selected ion of in the one-level mass spectrophotometry, measuring.Can specifically from the one-level mass spectrophotometry, choose and be used for possible fracture and further quality analysis because have the ion of specific mass-to-charge ratio, so be not brought in the second order ms analysis and therefore can not pollute the spectrogram that second order ms is analyzed from the unselected ion of one-level mass spectrophotometry.In addition, the linearity of mass spectrometric sensitivity and detecting device (for quantitative purpose) can be quite sane in this inferior quality scope.And the mass spectrometry art of present stage can allow in this mass range less than 1 daltonian baseline mass resolution.

Counterbalance (or connector) part:

The counterbalance (or connector) that can be used for the labelled reagent of embodiment of the present invention partly (uses abbreviation " LK " expression) sometimes will report that thing part and analyte or report thing partly couple together with reaction active groups, depend on whether the reaction with analyte takes place.Can select connector to produce neutrals (promptly experience neutrality is lost in mass spectrometer), wherein connect connector and report thing partly key (RL key) and the key (LA key) that is connected connector and analyte in mass spectrometer, rupture.Connector can be designed to when (comprising inferior fracture) inferior fracture take place, thereby only produce the neutral fragment of connector when applying the energy level that dissociates.Can design connector to produce one or more detectable fragments.

Connector part can comprise one or more heavy atom isotopes, thereby its mass compensation is given the gross mass difference between (labelled reagent analyte or that be used for labelled reagent group and/or kit that is used for every kind of mark of potpourri) report thing part.And the set gross mass (promptly making as a whole gross mass) of report thing/connector combination (promptly reporting thing/connector part) is for for the analyte of every kind of mark in the potpourri or can be identical for the labelled reagent of labelled reagent group and/or kit.More specifically, connector part can compensate the gross mass difference between the report thing part of analyte of mark in the different samples, the gross mass of wherein reporting the uniqueness of thing part is associated with the sample that the analyte of mark is originated, and the set gross mass of report thing/connector combination all is identical for the analyte of every kind in sample mixture mark, and no matter the sample that analyte is originated how.Like this, mix then when producing sample mixture when carrying out mark, the gross mass of the same analyte in two or more different samples can have same gross mass.

For example, the analyte of mark or be used for the labelled reagent group of labelled analyte and/or the labelled reagent of kit can be isomeric and/or isobaric.Therefore, if in mass spectrometer, from the initial mass analysis of sample mixture, select ion (taking from sample mixture) (i.e. the ion of Xuan Zeing) with specific mass-to-charge ratio, then from the same analyte of the different samples of formation sample mixture can represent with its in sample mixture concentration separately and/or measure proportional selected ion.Therefore, connector not only connects report thing and analyte, thereby it can also be used for compensating unique gross mass (with reference to Fig. 2 a/2b and 3a/3b) of reporting report thing/connector part of the analyte that thing difference quality partly is in harmonious proportion different sample marks.

Because connector can be used as the mass balance thing of report thing part, so the number of atom is big more in the connector, then the possible number of the different isomeric/isobaric labelled reagent of labelled reagent group and/or kit is also big more.In other words, the atom number that common connector comprises is big more, then the possible report thing of Cun Zaiing/connector combination is just many more, because thereby isotope can replace the isomeride that produces the connector part in most of positions of connector and/or measure the dystopy thing together, wherein said connector partly is used to compensate difference quality and so the unique isomerism and/or isobaric labelled reagent group of generation of report thing part.These different labelled reagent groups especially are fit to the multiple analysis of analyte in the same and/or different samples.

The total number of the labelled reagent in labelled reagent group and/or kit can be two, three, four, five, six, seven, eight, nine, ten, 11,12,13,14,15,16,17,18, nineteen, 20 or more.The diversity of the labelled reagent in group and/or kit only is subjected to reporting the atom number of thing with the connector part, replaces the isotopic available heavy atom isotope of light atom and the restriction of different synthetic configuration (wherein isotope can be inserted by synthetic).Yet as indicated in preamble, the basic initiation material of multiple isotope enrichment can be easily obtains from manufacturer such as CambridgeIsotope Laboratories and Isotec.The basic initiation material of these isotope enrichments can use in the building-up process of preparation isomerism and isobaric labelled reagent group, perhaps is used for producing the basic initiation material of the isotope enrichment of using in the building-up process of the isomeric and isobaric labelled reagent group of preparation.In this respect with lower banner: carried out more detailed argumentation under " the labelled reagent preparation method illustrates ".

Preparation is applicable to that some embodiment of the same amount dystopy labelled reagent of labelled reagent group discuss below in more detail.For example, the connector part can be represented by formula 1#:

Wherein by X ₁, X ₂, K, L, R ₁And R ₂The atom of representative or group are in the following more detailed description of having carried out.

Report thing/connector combination (promptly reporting thing/connector part):

Labelled reagent can comprise report thing part and the connector part that is connected to each other directly.As mentioned above, report thing/connector part can be identical on gross mass for each member in labelled reagent group and/or the kit.In addition, the key that connects report thing part and connector part applies at least a portion of the selected ion that ruptures when dissociating energy level through design with box lunch, reports the thing ion thereby partly discharge from connector part and/or connector/analyte.Therefore, can be in MS/MS analyzes the directly gross mass (in mass spectrometer, being detected than (being m/z)) and the intensity thereof of examining report thing ion as matter.

Report thing/connector part can be included in the various combinations of the identical or different heavy atom isotope of the various labelled reagents in labelled reagent group or the kit.In scientific literature, this is called as " coding " (" coding "), " isotope-coded " (" isotope coding ") sometimes or abbreviates " coding " (" encoding ") as.For example, Abersold etc. discloses isotope-coded affinity label (ICAT; Referring to WO00/11208).On the one hand, the reagent of Abersold etc. and the difference of labelled reagent of the present invention are that Abersold does not instruct the labelled reagent of two or more equals in quality, such as isomerism and/or isobaric labelled reagent.On the contrary, Abersold etc. has instructed " gently " of its labelled reagent and the form of " weight ".

In some embodiments, report thing and/or connector part can comprise the analyte that can be used for labelled reagent or mark and is fixed to atom or group on the carrier.Fixing can be direct or indirect.For example, in some embodiments, if reaction active groups (for example connector that can the disconnect) direct interaction of atom that combines with report thing and/or connector or group (for example reporting the alkyl amine substituting group of thing and/or connector) and carrier is fixed realizing, then can take place directly fixing.Comparatively speaking, fix realizing if for example will report reaction active groups interaction that the substituting group (for example reporting the alkyl amine substituting group of thing and/or connector) of thing and/or connector modifies (for example by biotinylation) and modification group and carrier (for example avidin or streptavidin), indirect securement then takes place.Therefore, the present invention relates to analyte wherein can with the embodiment of carrier-bound labelled reagent reaction, wherein every kind of carrier comprises unique labelled reagent, thereby different samples and the reaction of different carrier, and the present invention relates to wherein that every kind of different sample reacts with different labelled reagent and after this reaction product is fixed to embodiment on the identical or different carrier.Under each situation, sample mixture is usually all by utilizing mass spectrometer that the analyte of mark is obtained (referring to Fig. 5) from the carrier disconnection that is used to analyze.

Mass spectrometer/mass spectroscopy (MS):

Can use tandem mass spectrometer and other mass spectrometer of the ability that has selection and rupture molion to put into practice method of the present invention.Tandem mass spectrometer (and than the single-stage mass spectrometer on the back burner) has that mass-to-charge ratio (m/z) according to molion is selected and fracture molion and the ability that writes down resulting fragment (sub-fragment) ionic spectrum then.More specifically, can produce sub-fragment ions spectrum by selected ion being applied the energy level that dissociates (being collision induced dissociation (CID)).For example, can from the one-level mass spectrophotometry, select peptide, in the second order ms analysis, it be ruptured and analysis again corresponding to mark with specific m/z ratio.The representative instrument that can carry out such series connection quality analysis includes but not limited to magnetic four districts (magnetic four-sector) mass spectrometer, time-of-flight mass spectrometry instrument, triple quadrupole bar mass spectrometer, ion trap mass spectrometer and mixing quadrupole rod flight time (Q-TOF) mass spectrometer.

The mass spectrometer of these types can make up with various types of ionization sources, and described ionization source includes but not limited to electro-spray ionization (ESI) and substance assistant laser desorpted ionized (MALDI).Ionization source can be used for producing the charged species of first order mass spectrophotometry, and wherein analyte does not have fixing electric charge.Other mass spectroscopy instrument and fracture method comprise the post-source decay in the MALDI-MS instrument and use the high-energy CID of MALDI-TOF (flight time)-TOF MS.Summary recently about tandem mass spectrometer sees also: R.Aebersold and D.Goodlett, MassSpectrometry in Proteomics.Chem.Rev.101:269-295 (2001).

Fracture by the energy level that dissociates (Dissociative Energy Level):

As everyone knows, the fracture of key can be used as the result who occurs in the process in the mass spectrometer.And, can dissociate energy level and in mass spectrometer, cause bond rupture by applying to ion.For example, the energy level that dissociates can pass through collision induced dissociation (CID is also referred to as dissociating or CAD of collisional activation sometimes) and produces in mass spectrometer.Those of ordinary skill in the field of mass spectrometry will be understood that, other example technique that is used to apply the energy level that dissociates that causes fracture includes but not limited to photodissociation, electron capture dissociation (ECD), electron transfer dissociation (ETD, referring to Proc.Nat ' l Acad.Sci.USA. such as U.S. publication application No.2005-199804A1 and Syka, 9528-9533 (2004)) and spatial induction dissociate (SID) 101 (26):.For the purpose of explaining this instructions, the energy level that dissociates also can be thought the gas-phase chemical reaction that ruptures of causing that comprises any kind in mass spectrometer.

Fracture process by collision induced dissociation comprises by making the kinetic energy state of selected ion be increased to the point that bond rupture takes place with the inert gas collision.For example, kinetic energy can be by transferring in the collision pond with the collision of inert gas (such as nitrogen, helium or argon gas).The amount of the transferable kinetic energy of giving ion is proportional with the number of the gas molecule that allows to enter the collision pond.When having more gas molecule, more substantial kinetic energy is transferable to selected ion, when having less gas molecule, and transferable less kinetic energy.

Therefore, know that very the energy level that dissociates in the mass spectrometer can control.Generally acknowledge that also some key is more unstable than other key.The instability of the key in analyte or report thing/connector part depends on the character of analyte or report thing/connector part.Therefore, scalable is dissociated energy level so that analyte and/or label (for example reporting thing/connector combination) rupture in the mode that can measure.It will be understood by those skilled in the art that how mass spectrometric parts being carried out such routine regulates and realize the suitable energy level that dissociates, thereby the ion fragmentation of the analyte of the mark of at least a portion is become Ionized report thing part and sub-fragment ions.

For example, dissociation energy can be imposed on the ion of selection/separation from the one-level mass spectrophotometry.In tandem mass spectrometer, the energy level that dissociates can be imposed on the ion of extraction, then it is transferred to the second order ms analyser.The ion of selecting can have selected mass-to-charge ratio.Mass-to-charge ratio can be in the scope by the mass-to-charge ratio that mass spectrometric character determined.When using collision induced dissociation, by making ion, ion can be transferred to the second order ms analyser from the one-level mass spectrometer by colliding the pond, produce fragment ions thereby in described collision pond, can apply the energy level that dissociates.For example, the example that is sent to the ion that the second order ms analyser is used to analyze comprises the sub-fragment ions of residual (not fracture) some of selected ion or the analyte of a part and report thing ion (signal ion) and mark.

Analyte determination by the computer-Aided database analysis:

In some embodiments, can be based on daughter ion (daughter-ion) fracture mode determination and analysis thing, described daughter ion fracture mode is analyzed by comparison computer assisted and known or " theory " analyte spectra.The sub-fragment ions spectrum of the peptide ion that for example, ruptures under low-yield CID can be thought the summation of many discrete fracture incidents.Sub-fragment ions has been distinguished in common name, according to the fracture amido link and after bond rupture still charged fragments of peptides (Reopstorff etc., Biomed.Mass Spectrom., 11:601 (1988)).The electric charge that keeps in the terminal side of the N of the amido link of easy fracture causes forming b type ion.If the terminal side of the C of the amido link of fracture keeps electric charge, then fragment ions is called y type ion.Except b type and y type ion, the CID mass spectrum can comprise other diagnostic fragment ion (sub-fragment ions).These comprise by lose ammonia from glutamic acid, lysine and arginine neutrality (17amu) or from the amino acid (as serine and threonine) that contains hydroxyl loses the water (ion that 18amu) produces.Observed under low-yield CID condition some amino acid than the easier fracture of other amino acid.For the peptide that contains proline or asparagicacid residue, this is especially apparent, for aspartyl-proline key even (Mak, M. etc., Rapid Commun.MassSpectrom., 12:837-842 (1998)) all the more so.Therefore, than the peptide bond between all other amino acid dimers combination, Z " '-pro dimer or Z " '-dimeric peptide bond of asp (wherein Z " ' be any neutral amino acid, pro is a proline, asp is an aspartic acid) tend to more unstable.

Therefore, for peptide and protein example, low-yield CID spectrum comprises overlapping b series and the serial ion of y, loses the sequence-specific information of the redundancy of ion from the interior segments ion of same peptide and imonium ion (immonium) and other neutrality.Although it is challenging and time-consuming that such CID spectrum is resolved with the amino acid sequence of from the beginning assembling parent peptide, but still can carry out.Recent advances about computer assisted de novo sequencing method is described in Huang, Y., Ross, P, Smirnov, I, Martin, S. and Pappin, D.2003, Proceedings of6th International Symposium on MS in Health and Life Sciences, Aug24-28,2003, among the San Francisco CA.Identifying that the most significant progress aspect the peptide sequence is the progress of computerized algorithm, described algorithm makes peptide CID spectrum be associated with peptide sequence in being present in protein and dna sequence data storehouse.These methods are by program such as SEQUEST (Eng, J.Am.Soc.Mass Spectrom. such as J., 5:976-989 (1994)) and MASCOT (Perkins, Electrophoresis such as D., 20:3551-3567 (1999)) and illustrate.

In brief, the peptide CID of experiment spectrum (MS/MS spectrum) is complementary with " theoretical property " the sub-fragment ions spectrum that is produced from the peptide sequence that derives from protein or genomic sequence data storehouse by computing machine or is relevant.This coupling or relevance are based in the forecast quality of MS/MS pattern neutron fragment ions and the similarity between the detected quality.Give possible matching or relevance score according to this experimental degree that is consistent with " theoretical property " fracture mode.Constraint condition for the database retrieval of given peptide ammino acid sequence is that resolving ability is so arranged, to such an extent as to single peptide CID spectrum can be enough to identify any given protein in the database of whole genome or expressed sequence tag (EST).Other summary sees also: Yates, J.R.Trends, Genetics, 16:5-8 (2000) and Yates, J.R., Electrophoresis 19:893-900 (1998).

Therefore, the sub-fragment ions analysis of MS/MS spectrum not only be can be used for measuring the analyte of labelled analyte, also can be used for measuring the analyte that described determined analyte is originated.For example, the evaluation of peptide can be used for measuring protein in MS/MS analyzes, and described peptide ruptures from described protein as the protein of enzymic digestion.Can predict, this analysis can be used for other analyte, such as nucleic acid, lipid and/or steroids.

RL key and LA key:

Key between the atom of report thing part and the atom of connector part is the RL key.Key between the atom of connector part and the atom of analyte is the LA key.In some embodiments, when being applied in when dissociating energy level, RL key and LA key in the selected ion of at least a portion all can rupture.Therefore, in some embodiments, can in mass spectrometer, regulate the energy level that dissociates, so that RL key in the selected ion of at least a portion of labelled analyte and LA key all rupture.

The fracture of RL key discharges report thing part from analyte, so that can be independent of analyte determination report thing ion.The fracture of LA key discharges report thing/connector part from analyte, perhaps discharges connector from analyte, and this depends on whether the RL key ruptures.In some embodiments, the comparable LA key of RL key is more unstable.In some embodiments, the comparable RL key of LA key is more unstable.In some embodiments, RL key and LA key can have same relative instability.In brief, in various embodiments of the present invention, design makes the RL bond rupture, thereby discharges report thing ion, but the LA key may rupture or not rupture.

In some embodiments, when interested analyte is protein or peptide, the relative instability of acid amides (peptide) key of scalable RL key and LA key.Than typical acid amides (peptide) key, RL key, LA key or RL key and LA key all can be more unstable, stable or more stable equally.For example, under the condition of dissociation energy, with Z " '-pro dimer or Z " '-asp dimer compares, RL key and/or LA key can be not easy to fracture, wherein Z " ' be any natural amino acid, pro is a proline, asp is an aspartic acid.In some embodiments, the RL key can rupture under the about identical energy level that dissociates of typical amido link with the LA key.In some embodiments, compare with typical amido link, RL key and LA key can rupture under the higher energy level that dissociates.

In some embodiments, can have RL key and LA key, so that the fracture of RL key causes the fracture of LA key, and vice versa.In the method, RL key and LA key be fracture simultaneously basically, so that do not have the analyte of significant quantity or its sub-fragment ions to comprise the part label." analyte of significant quantity " means the analyte that is less than 25%, preferably is less than 10% part mark and can (for example in MS/MS analyzes) be measured in mass spectrometer.

In some embodiments, because in mass spectrometer, can have clearly boundary between labeled fragment of (for example in MS/MS analyzes) analyte and the unmarked fragment, so this feature can be simplified the evaluation from the analyte of the computer-aided analysis of sub-fragment ions spectrum, because do not need the compensation of label residue is imposed on the Mass Calculation of the analysis of the sub-fragment ions that is used for analyte.In addition, because the fragment ions of analyte is all mark or unlabelled (but not being the part mark) in certain embodiments, disperse so seldom exist or do not exist by the caused quality of the isotopic distribution of crossing over breaking bonds, thereby can there be such situation: cause the fracture of the analyte of mark owing to using the energy level that dissociates, cause isotope to be present in each side of single easy scission of link of the analyte of part mark.

The mark of analyte:

As previously mentioned, the functional group that can be by making analyte and the reaction active groups reaction of labelled reagent and labelled analyte.The functional group of analyte can be a kind of in electrophilic group or the nucleophilic group, and the functional group of labelled reagent can be the another kind in electrophilic group or the nucleophilic group.Parent electric thing and nucleophilic thing can react to form covalently bound between analyte and labelled reagent.

Labeled reactant can take place in solution.In some embodiments, a kind of in analyte or the labelled reagent can be carrier-bound.Labeled reactant can carry out under aqueous conditions sometimes.Can select aqueous conditions for mark biomolecule (as protein, peptide and/or nucleic acid).Labeled reactant can carry out in the potpourri of organic solvent or organic solvent sometimes.Can select the organic solvent of micromolecular analyte.Can in broad range, make the potpourri of water and one or more organic solvents.For example, can prepare and make water and about 5% to the solution of one or more organic solvents (v/v) of about 95% with labelled analyte.In some embodiments, can prepare and make water and about 50% to the solution of one or more organic solvents (v/v) of about 95% with labelled analyte.In some embodiments, can prepare and make water and about 65% to the solution of one or more organic solvents (v/v) of about 80% with labelled analyte.The example of the indefiniteness of organic solvent comprises N, and N '-dimethyl formamide (DMF), acetonitrile (ACN), N-crassitude (NMP) and alcohols are such as methyl alcohol, ethanol, propyl alcohol and/or butanols.Those skilled in the art utilize to be no more than knowledge and content disclosed herein and the conventional experiment that can know this area according to the character of labelled reagent and the character of analyte, can determine to promote the suitable solvent condition of analyte mark.

When carrying out labeled reactant, scalable pH.PH can be in the scope of 4-10.PH also can be beyond this scope.Usually, can be by adding the alkalescence that non-nucleophilicity organic base is regulated non-aqueous reaction.The non-limiting example of suitable alkali comprises N-methylmorpholine, triethylamine and N, the N-diisopropylethylamine.Perhaps, can utilize biological buffer (such as the N-[2-hydroxyethyl] piperazine-N '-[2-ethanesulfonic acid] (HEPES) or 4-morpholino b acid (MES)) or inorganic buffer agent (such as sodium carbonate and/or sodium bicarbonate) regulate the pH of aqueous solvent.Because one of reactive group can be electrophilic at least, may be desirable so selection does not contain the buffering agent of any nucleophilic group.Under the condition of using normal experiment, those skilled in the art can be identified for other buffering agent of the pH of aignment mark reaction, thereby promote the mark of usage flag reagent to analyte.Therefore, those skilled in the art can utilize the experiment that is no more than content disclosed herein and routine to determine suitable solvent and pH condition, thereby promote the analyte mark according to the character of labelled reagent and the character of analyte.

Sample preparation:

In certain embodiments of the invention, can and handle sample before one or more analytes of mark afterwards.Processing can promote the mark to described one or more analytes.Described processing can promote the analysis to sample component.Processing can be simplified the operation to sample.Processing can promote aforesaid two or more multinomial.

For example, available enzyme or chemicals are handled sample.Enzyme can be other a enzyme of proteinase (with degrade proteins and peptide), nuclease (with degraded nucleic acid) or some.Can select enzyme to have lucky foreseeable degraded mode.Two or more proteinase and/or two or more nucleases can also be used together, or use with other enzyme, thus the degraded sample component.

For example, proteolytic enzyme trypsase is a kind of serine protease, thereby the peptide bond between its broken filaments propylhomoserin or arginine and the nonspecific amino acid produces the peptide that comprises amine end (N end) and serine or arginine carbonyl end amino acid (C end).In the method, the peptide that ruptures from protein is predictable, and it can become the existence of its protein of originating and/or the indication of amount from existence in the sample of trypsinization thing and/or amount.In addition, the unhindered amina end of peptide can be the good nucleophilic thing that promotes the mark of peptide.Other exemplary proteolytic enzyme comprises papain, pepsin, ArgC, LysC, V8 proteinase, AspN, pronase, chymotrypsin and carboxypeptidase (for example Carboxypeptidase A, B, C etc.).

For example, when with proteinase (as trypsase) digestion, protein (for example albumen g) may produce three kinds of peptides (for example peptide B, C and D).Therefore, used the sample that proteolytic enzyme (such as trypsase) hydrolysis is crossed and affirmation contains peptide B, C and D when analyzing to be considered to comprise at first albumen g.The amount of peptide B, C and D also will be associated with the amount of albumen g in the sample that digests.In the method, about among peptide B, C in the sample (or its part) and the D one or more evaluation and/or quantitative any mensuration all can be used to identify and/or the quantitative protein in the original sample (or its part).

Because the activity of some enzyme is predictable, so the sequence of the peptide that is produced by the protein degradation of known array also is predictable.According to this information, can produce " theoretic " peptide information.Therefore, in computer-aided analysis, the mensuration of " theoretic " fragments of peptides be can be used for measuring one or more peptides in one or more unknown samples or protein (for example referring to the part of heading for " analyte determination of analyzing by computer-Aided database ") from the sub-fragment ions (as mentioned above) of the mass spectrophotometry of actual sample.

In some embodiments, sample preparation can comprise the processing to the precursor of one or more analytes to be marked.For example, if one or more analytes to be marked are the peptides that come from the protein of digestion, and selected marker reagent concerning this example is so that amine groups (for example the N-α-amine groups of lysine and the N-ε-amine groups) reaction of itself and peptide or peptide analysis thing can promote the mode of labeled reactant to handle the protein of sample (analyte precursor molecule).In this example, available reductive agent (for example three [2-carboxyethyl] phosphines (TCEP)) is with the protein reduction, then by sealing with the reaction of sealer (for example methane thiosulfonic acid methyl esters (MMTS)).In the method, the thiol group of protein is closed and so the amine of not interference analysis thing and the labeled reactant between the labelled reagent.

It will be understood to those of skill in the art that and utilize the reagent be easy to get and, can carry out processing some other precursor molecule by the adjustable scheme of normal experiment.Can accurately select reagent and condition according to the character of analyte to be marked and labelled reagent.

In some embodiments, sample preparation can comprise analyte or analyte precursor are fixed on the solid carrier, no matter and whether use the labelled reagent mark.Fix and to comprise Covalent Immobilization and absorption and other non-covalent fixing means (for example static is fixed).In some embodiments, the fixing complicacy that can help to reduce product.In some embodiments, fixingly can help the analyte mark.In some embodiments, fixingly can help body tag before the analyte.In some embodiments, fixing can helping, carried out selected marker to the part of sample component with some character (for example, they have or lack the halfcystine part).In some embodiments, fixingly can help purifying.Fixing can help aforementioned in every two or multinomial.

Comprise the separation of sample separation potpourri:

In some embodiments, may relate to separation to the sample of labelled analyte or the processing of sample mixture.Can carry out one or more separation to mark or unlabelled analyte, mark or unlabelled analyte precursor or its part.Can carry out one or more separation to one or more parts of the other products that derives from solid-phase capture thing or detachment process.Can be to aforementioned two or multinomial separation the in every.

For example, can prepare the sample mixture that comprises different labelled analytes from different samples.Each label that " not isolabeling " means difference each other comprises identifiable peculiar property (for example each label comprises unique report thing part, and the report thing part of described uniqueness produces unique " signal ion " in MS/MS analyzes).Be the analytic sample potpourri, the component of separable described sample mixture is also carried out quality analysis to the only part of described sample mixture.In the method, the complicacy of analyte can fully be reduced, because can carry out quality analysis separately to the analyte that separates, thereby has increased Sensitivity of Analytical Method.Certainly can carry out the replicate analysis one or many to one or more other parts of sample mixture, thereby can analyze all parts of described sample mixture.

Separation condition (under the described conditions not the same analyte of isolabeling with proportional concentration of its abundance in sample mixture or amount under co-elute) can be used to measure the amount of every kind of labelled analyte in every kind of sample (described sample comprises sample mixture), prerequisite is that the amount that every kind of sample is added in the described sample mixture is known.Therefore, in some embodiments, the separation of sample mixture can be simplified analysis, and keeps in the signal measured in the quality analysis (for example MS/MS analyze) and the sample mixture the not correlativity between the amount of the analyte of isolabeling simultaneously.

Can separate by chromatography.For example, can use liquid chromatography/mass spectrometry (LC/MS) to realize sample separation and quality analysis.And, can use any suitable chromatography separating method to separate interested analyte.For example, chromatographic resolution can be normal phase chromatography, reversed phase chromatography, ion-exchange chromatography (being anion-exchange chromatography or cation-exchange chromatography), size exclusion chromatograph method or affinity chromatography.

Separation can be carried out under electrophoresis.The non-limiting example of spendable electrophoretic separation technique includes but not limited to that one dimension electrophoretic separation, two dimensional electrophoresis separate and/or capillary electrophoresis separation.

Can use isobaric labelled reagent or reagent set to come the analyte of mark sample.When carrying out separating step, isobaric labelled reagent is particularly useful, because the same amount dystopy label of labelled reagent group structurally can not be distinguished (and can not distinguish by gross mass) before the report thing is removed in the analyte fracture.Therefore, all analytes with the same composition of different isobaric label marks can both carry out chromatographic resolution (being co-elute) in the identical mode of strictness.Because they are undistinguishable structurally, the eluate of detachment process can comprise a certain amount of isobaric labelled analyte, and the amount of the labelled analyte in described labelled analyte and the sample mixture is proportional.And, according to the knowledge (for the umber of the preparation sample that adds of sample mixture and other optional member (for example calibration standard thing)) of preparation sample mixture, the amount of the labelled analyte in the amount that may make labelled analyte in the sample mixture and the sample that labelled analyte is originated is relevant.

Labelled reagent also can be isomeric.Although isomeride can pass through chromatographic resolution sometimes, but the situation that existence condition relies on, wherein can carry out detachment process with co-elute all by the same analyte of isolabeling not, wherein be present in the amount of the whole labelled analytes in the eluate and its in sample mixture concentration and/or measure proportional.

The relative quantification of analyte and absolute quantitation:

In some embodiments, in the sample mixture not the same analyte of isolabeling to carry out relative quantification be possible.For example, relative quantity by the report thing ion (being the signal ion) measured with quality analysis (for example detected labelled analyte is selected and is used for the second order ms analysis in to the one-level mass spectrophotometry) (for example the area at Bao Gao peak and/or highly) compares, and it is possible that the same analyte of isolabeling is not carried out relative quantification.In other words, can be at every kind of report thing ion with under the information of used specific sample is associated with the situation that produces sample mixture, report thing ion promptly is the relative quantity of analyte in the sample mixture with respect to the relative quantity of detected other report thing ion in mass spectrophotometry.Under the known situation of the constituent that forms sample mixture, can be based on the relative quantity of the report thing ion of the detected labelled analyte that is used to have selected mass-to-charge ratio, the relative quantity inverse of analyte that will be used for preparing every kind of sample of sample mixture comes out.For detected all different labelled analytes in the one-level mass spectrophotometry, all can repeat this process.In the method, can measure the relative quantity (often showing) of every kind of reactivity analyte of every kind of different samples that are used for preparing sample mixture with concentration and/or scale.

In other embodiments, can determine the absolute quantitation of analyte.For these embodiments, one or more that can add known quantity in sample mixture are the analyte of isolabeling (one or more calibration standard things) not.The calibration standard thing can be the predictive analysis thing with isomerism and/or isobaric label or label group (analyte that is used for the mark sample mixture) mark, and precondition is that the report thing part of described calibration standard thing compares with any sample that is used to form sample mixture is unique.Utilize with sample mixture in the correlativity of the relative quantity of one or more report thing ions of the analyte of isolabeling not, in case determine the relative quantity of described relatively one or more calibration standard things of report thing ion, then can be with reference to the amount that joins the calibration standard thing in the sample mixture all absolute magnitudes of the analyte of isolabeling (often showing) not in the calculation sample potpourri with concentration and/or scale.In the method, also can determine every kind of not absolute magnitude of the analyte of isolabeling (, in the sample in its source, having the calibration standard thing) based on the knowledge of the method for preparing sample mixture for it.

Although aforementioned content is arranged, when needed, also can make the correction of report thing ion (signal ion) intensity at any isotopic abundance natural or artificial generation in the report thing part.The isotopic abundance that impurity in the several different methods correction report thing part signal ion is arranged.The example of such modification method is found in the U.S. Patent No. 7,105,806 that disclosed common unsettled and total name is called " Method andApparatus For De-Convoluting A Convoluted Spectrum ".Basically, can utilize mathematical formulae and calculating, calculate isotope bunch relevant the adding of determining with single marking reagent mass peak (up-mass peak) and subtract mass peak (down mass peak) by deconvolution to the flatung spectrum of the overlapping isotope of labelled reagent bunch.No matter how value determines that careful more aspect the intensity of accurate quantitative every kind of report thing ion (being the signal ion), the relative quantification of analyte and absolute quantitation are just accurate more in the sample of source.

Proteomic analysis:

Embodiment of the present invention can be used for complex analyses because can utilize the quality analysis technology, with fast and the mode that repeats with multipleization of sample (multiplexed), analyze and analyze again.For example, can carry out the sample mixture analysis, to determine the amount of one or more analytes in one or more samples.Can be at the amount (often showing) of one or more analytes of sample determination that sample mixture comprised with concentration and/or scale.Because can carry out sample preparation and quality analysis fast, thus these methods can repeat repeatedly so that relative quantity and/or the absolute magnitude of multiple different labelled analytes that can the working sample potpourri in the sample of analyte source.

Can use a kind of application of so quick multiple analysis (multiplex analysis) is in the Proteomic analysis field.Proteomics can be regarded a kind of experimental method of describing coded message in the genome sequence aspect structure, function and the regulation and control at bioprocess as.This can or organize the systematicness analysis of expressed gross protein composition to realize by pair cell.The mass spectrum that is used in combination with the embodiment of method of the present invention, potpourri, kit and/or composition be used for a kind of of this comprehensive protein analysis may instrument.

For example, utilize 9 kinds with amount dystopy labelled reagent group, 9 time points in can obtaining to test are to measure for example based on rise or the downward modulation of auxocyte to the protein expression of replying of particular stimulation thing.Also can carry out less time point but introduce one or more testers.Same multiple experiment is doubled or become three times to carry out.Under all scenario, all can in single multiple experiment, measure randomly the rise or the downward modulation of the protein expression that repeats with respect to one or more optional testers and/or sample.In addition, because the processing of sample mixture is parallel carrying out,, thereby do not need in the compensation of making aspect scheme or the experiment condition subtle change so the result is directly comparable.Therefore, these isobaric labelled reagents can with experimental analysis include but not limited to that (PTM) measured in experiment, biomarker analysis, multiplexed protein matter group analysis, multidimensional protein identification techniques experiment (mudpit experiments), compatibility pull-downs experiment (affinitypull-downs), the posttranslational modification of time course and (multiple controlexperiment) tested in multiple control.

II. composition

In some embodiments, the present invention relates to the composition that comprises its salt form and/or hydrate forms by formula I representative:

Wherein, group Y-J can be any report thing group.The character of suitable report thing group was described the front in this article.The character of suitable report thing group also in U.S. publication application No.US 2004-0219685-A1, was especially described in the 41-47 section.

For example, the report thing can comprise and can be substituted or unsubstituted 5,6 or 7 yuan of heterocycles, and described heterocycle can randomly can be disconnected to be connected on the carrier, and wherein said heterocycle comprises by covalent bond and is connected at least one theheterocyclic nitrogen atom on the group J.Group J can be by formula-CJ ' ₂The replacement of-representative or unsubstituted methylene, wherein each J ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₃,-OR ₃,-SR ₃,-R ₃' OR ₃Or-R ₃' SR ₃Group K can be by formula-(CK ' ₂) _n-or-((CK ' ₂) _m-X ₃-(CK ' ₂) _m) _pThe group of-representative, wherein n is 2～10 integer, each m is 1～5 integer independently of one another, p is 1～4 integer, each K ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₄,-OR ₄,-SR ₄,-R ₄' OR ₄Or-R ₄' SR ₄Group L can be by formula-(CL ' ₂) _q-or-((CL ' ₂) _m-X ₃-(CL ' ₂) _m) _pThe group of-representative, wherein q is 1～10 integer, each m is 1～5 integer independently of one another, p is 1～4 integer, each L ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₅,-OR ₅,-SR ₅,-R ₅' OR ₅Or-R ₅' SR ₅About radicals R ₁And R ₂, or (1) R ₁Be hydrogen, deuterium or R ₆, R ₂Be hydrogen, deuterium or R ₇(2) R ₁And R ₂Be together by formula-(CR ' ₂) _q-or-((CR ' ₂) _m-X ₃-(CR ' ₂) _m) _pThe group of-representative, described group have formed the ring of two nitrogen-atoms of bridge joint, and wherein q is 1～10 integer, and each m is 1～5 integer independently of one another, and p is 1～4 integer, each R ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₆,-OR ₆,-SR ₆,-R ₆' OR ₆Or-R ₆' SR ₆Atom or radicals X ₁Can be=O ,=S ,=NH or=NR ₇, each X ₂Can be independently of one another=O or=S, each X ₃Can be independently of one another-O-or-S-.Group Z can be-OH ,-SH ,-O ^-V ⁺,-S ^-V ⁺, reaction active groups, the leaving group of reaction active groups or the analyte of covalent bonding; Wherein, V ⁺It is positively charged gegenion.Each R ₃, R ₄, R ₅, R ₆And/or R ₇Can be alkyl, thiazolinyl, alkynyl, aryl, heteroaryl or aryl alkyl independently of one another.Each R for example ₃, R ₄, R ₅, R ₆And/or R ₇Can be methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, isobutyl, sec-butyl or the tert-butyl group independently of one another.Each R ₃', R ₄', R ₅' and/or R ₆' can be alkylidene, alkenylene, alkynylene, arlydene or alkylidene aryl independently of one another.For example, each R ₃', R ₄', R ₅' and/or R ₆' can be methylene, ethylidene, propylidene, cyclopropylidene, inferior normal-butyl, inferior cyclobutyl, inferior n-pentyl, cyclopentylene, inferior n-hexyl or cyclohexylidene independently of one another.

Described composition can be isotope enrichment (i.e. a coding).Described composition can be isotope enrichment to comprise one or more heavy atom isotopes.Described composition can be isotope enrichment to comprise two or more heavy atom isotopes.Described composition can be isotope enrichment to comprise three kinds or more kinds of heavy atom isotope.Described composition can be isotope enrichment to comprise four kinds or more kinds of heavy atom isotope.

Described 5,6 or 7 yuan of heterocycles can be any 5, the 6 or 7 yuan of heterocycles that comprise at least one nitrogen-atoms, and described group J can be covalently bound with described nitrogen-atoms.For example, described heterocycle can be to replace or unsubstituted morpholine, piperidines or piperazine.Possible substituting group " definition " part was in front described, and wherein said heterocycle can comprise one or more described substituting groups.For example, substituting group can be hydrogen, deuterium, methyl ,-C (H) ₂D ,-C (H) D ₂,-CD ₃Or other alkyl.Described substituting group can with the heteroatoms of described ring bonding mutually.For example, described heterocycle can be a N methyl piperazine.Described heterocycle can be an aromaticity or nonaromatic.

In some embodiments, report thing part can be disconnected to be connected on the carrier.Various carriers are well known in the art.For example, the various carriers that comprise trityl part are commercial sell or (trityl chlorine carriers (trityl-Cl) or 2-chlorine trityl chlorine carrier) for example that can make.With reference to Fig. 5, for example understand an embodiment of carrier-bound labelled reagent.With reference to Fig. 5, thereby the available analyses thing is handled the analyte that carrier makes mark.As illustrational, usable acid processing carrier is used for analyzing with the analyte that discharges mark then.

Therefore, in some embodiments, described 5,6 or 7 yuan of heterocycles can comprise atom or the group that promotes that described heterocycle combines with disconnecting of suitable carriers.For example, described group can be alkylidene, alkenylene, alkynylene, arlydene or the alkylidene aryl group that contains amino, hydroxyl or mercapto.Described atom can be the secondary nitrogen on the piperazine ring.Discussion about exemplary diethylenediamine compound and preparation method thereof is found among the laid-open U.S. Patents application US 2004-0219685 A1.For example, can make described carrier-bound N-alkyl piperazine acetic acid compound and diamine reactant, thereby,, be possible wherein with amount dystopy coding based on the character of reactant then by forming the carrier-bound compound that can be used as labelled reagent with diacid reactant.

Refer again to formula I, group Y-J-(no matter whether it can be disconnected to be connected with carrier) can form report thing part.Described report thing part can comprise at least one with amount dystopy enrichment positions.Described report thing part can comprise at least two with amount dystopy enrichment positions.Described report thing part can comprise 3,4,5,6,7,8,9,10,11,12,13,14,15,16 or 17 or more a plurality of with amount dystopy enrichment positions.For example, Fig. 6 a understands that for example comprising 21 N methyl piperazines with amount dystopy enrichment positions reports things.

Described report thing part can comprise fixed charge or be ionogenic in mass spectrometer.For example, the compound that contains basic group (for example amine groups) is easy to protonated to introduce electric charge, the compound (for example hydroxy-acid group) that contains acidic-group then is easy to take off proton and introduces electric charge (referring to Roth, Kenneth etc. " Charge Derivatization of Peptides for Analysis byMass Spectrometry ", Mass Spectrometry Reviews, 17:255-274 (1998)).

Counterbalance (connector) part can form by the group by formula I# representative:

R wherein ₁, R ₂, X ₁, X ₂, K and L as above define.Described counterbalance part can comprise at least one with amount dystopy enrichment positions.Described counterbalance part can comprise at least two with amount dystopy enrichment positions.Described counterbalance part can comprise 3,4,5,6,7,8,9,10,11,12,13,14,15,16 or 17 or more a plurality of with amount dystopy enrichment positions.For example, for example clear counterbalance parts that comprise 28 with amount dystopy enrichment positions of Fig. 6 b, wherein the quality total increment can reach 31 dalton.

In some embodiments, described composition can comprise its salt form and/or hydrate forms by formula II representative:

Wherein W is ortho position, a position or the contraposition that replaces the atom of at least one M group of hexa-member heterocycle or group and be positioned at the nitrogen of described hexatomic ring.Group W can be-N (H)-,-N (R ")-,-N (R " ')-,-P (R ")-,-P (R " ')-,-O-or-S-.If elect as-N (R " ')-or-P (R " ')-, then this group can be used for described composition and carrier be can be disconnected to be connected.Each remaining group M can be-CM ' independently of one another ₂-, wherein each M ' can be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₈,-OR ₈,-SR ₈,-R ₈' OR ₈Or-R ₈' SR ₈Group J, K, L, X ₁, X ₂, R ₁, R ₂Define with Z such as front.Each R " can be alkyl, thiazolinyl, alkynyl, aryl, heteroaryl or aryl alkyl independently of one another, each R " ' can be H ₂N-R ₉'-, H (R ₁₀) N-R ₉'-, (R ₁₀) ₂N-R ₉'-, HO-R ₉'-, HS-R ₉'-or described compound be can be disconnected to the disconnected connector that couples together with carrier.Each R ₈And/or R ₁₀Can be alkyl, thiazolinyl, alkynyl, aryl, heteroaryl or aryl alkyl independently of one another, R ₉' can be alkylidene, alkenylene, alkynylene, arlydene or alkylidene aryl independently of one another.

In some embodiments, described composition can comprise its salt form and/or hydrate forms by the formula III representative:

Wherein s can be 1～5 integer, and t can be 1～10 integer.Radicals R ₁, R ₂Define with Z such as front.Atom and radicals R ₁₁Can be hydrogen, deuterium, methyl ,-C (H) ₂D ,-C (H) D ₂,-CD ₃, other alkyl or-R " ', wherein R " ' define as the front.For example, composition as described in one of can be from the compound V-XIII as shown in Fig. 2 a and 2b selecting.

In some embodiments, described composition can comprise its salt form and/or hydrate forms by formula IV representative:

Wherein, t, R ₁₁Define with Z such as front.For example, composition as described in one of can be from the compounds X V-XXIII as shown in Fig. 3 a and 3b selecting.

As described, described composition can its salt form and/or hydrate forms and existing.Whether described composition exists the condition that depends on that usually substituent character and number and composition are existing and/or separate as salt form.As everyone knows, protonated by can make basic group (such as amine) with acid treatment, thus the salt of formation amine.For example, the labelled reagent that contains piperazine can be used as single tfa salt, single HCl salt, two tfa salts or two HCl salt and obtains (referring to for example U.S. Patent Application Publication No.US 2005-0148771 A1).Also well-known, form carboxylate by can make acidic-group (such as carboxylic acid) take off proton with alkali treatment.The same, for example, can make carboxylic acid-OH or thiocarboxylic acid-SH takes off proton and formation-O respectively ^-V ⁺With-S ^-V ⁺, V wherein ⁺Be alkaline gegenion (Li for example ⁺, Na ⁺, K ⁺, Rb ⁺, Cs ⁺Or NH ₄ ⁺).Also well-known, the compound that comprises basic group (such as amine) and acidic-group (such as carboxylic acid) can exist by zwitterionic form.All these all are regarded as salt form, and the ionization state of these functional groups of composition will depend on the pH of its existing any solution, if when perhaps separating, then depend on the pH of any solution that is separated.Those of ordinary skills are bound to understand, and how only to utilize the state of charge and the character of any gegenion salt form of normal experiment method and content control composition disclosed herein disclosed herein.

Whether there is the condition that also will depend on its existence or separation in composition as hydrate.Hydrate only comprises the hydrone of one or more complexings.The present invention relates to any possible hydrate forms.

As mentioned above, group Z can with the analyte covalent bonding.Described analyte can make by making the reaction of analyte and labelled reagent.Described analyte can be any analyte.For example, group Z is peptide or protein.

Group Z can be foregoing reaction active groups.Therefore, in some embodiments, described compound can be to be selected from Compound I as described below '-any labelled reagent among XIII ' or the XV '-XXIII '.In some embodiments, described composition can be a labelled reagent, such as shown in compound 80 '-87 ' among Figure 25 a and the 25b those.

Group Z can also be-OH ,-SH ,-O ^-V ⁺Or-S ^-V ⁺Can be with described group activation, thus the leaving group of reaction active groups produced.The group of described activation is often referred to the hydroxy-acid group of activation.For example, can use foregoing water-soluble carbodiimide EDC with hydroxy-acid group (COOH)-OH base in-situ activation.

Group Z can also be the leaving group of reaction active groups (such as the hydroxy-acid group or the thiocarboxylic acid group (for example active ester) of activation).For example, the leaving group of described reaction active groups can be alcohol or the mercaptan leaving group of foregoing formula 7-19.Described active ester can be the N-hydroxy-succinamide ester (promptly described leaving group be compound 7 and X ' be-situation of O-under).As previously mentioned, can make nucleophilic group (such as the amino group) reaction of active ester and analyte, thereby form the analyte of mark.Therefore, such compound is a labelled reagent.

Described labelled reagent can be isomeric and/or isobaric.Other character of described labelled reagent is disclosed.For example, described labelled reagent can be used for the multiple analysis of one or more analytes in same sample or two or more the different samples.

Described labelled reagent can be isotope enrichment (i.e. a coding).Described labelled reagent can be isotope enrichment to comprise one or more heavy atom isotopes.Described labelled reagent can be isotope enrichment to comprise two or more heavy atom isotopes.Described labelled reagent can be isotope enrichment to comprise three kinds or more kinds of heavy atom isotope.Described labelled reagent can be isotope enrichment to comprise four kinds or more kinds of heavy atom isotope.Described labelled reagent can be isotope enrichment to comprise five kinds or more kinds of heavy atom isotope.Described labelled reagent can be isotope enrichment to comprise six kinds or more kinds of heavy atom isotope.Described labelled reagent can be isotope enrichment to comprise seven kinds or more kinds of heavy atom isotope.Described labelled reagent can be isotope enrichment to comprise eight kinds or more kinds of heavy atom isotope.Described labelled reagent can be isotope enrichment to comprise nine kinds or more kinds of heavy atom isotope.

In some embodiments, composition can be the calibration standard thing of mark.As described herein, the calibration standard thing of known quantity can be joined in the potpourri to promote the absolute quantitation analysis of interested analyte.Therefore, in some embodiments, the invention still further relates to the analyte of using isomerism and/or isobaric labelled reagent mark, such as interested peptide.Therefore, the calibration standard thing of described mark can be to use any analyte of labelled reagent mark as described herein.Usually, described labelled reagent can be selected from isomerism and/or isobaric labelled reagent group, so that it comprises than the report thing of the uniqueness of the labelled reagent that is used for one or more sample of interest of mark.

In some embodiments, the labeled analysis compositions is optional from Compound I described below "-XIII " or XV "-XXIII " in any one.In some embodiments, composition can be the analyte of mark, such as among Figure 26 a-26b 80 "-87 " shown in those.

In some embodiments, described composition can be a fragment ions.For example in some embodiments, described composition can be the fragment ions that is present in after the analyte molecule fracture of mark in the mass spectrometer.For example, the compound 80 shown in the optional Figure 26 freely of the analyte molecule of the mark of described generation fragment ions a-26b "-87 ".Therefore, described fragment ions can have following molecular formula: ¹³C ₆H ₁₃ ¹⁵N ₂ ⁺, ¹³C ₄C ₂H ₁₃ ¹⁵N ₂ ⁺, ¹³C ₃C ₃H ₁₃ ¹⁵N ₂ ⁺, ¹³C ₃C ₃H ₁₃ ¹⁵NN ⁺, ¹³C ₂C ₄H ₁₃ ¹⁵NN ⁺, ¹³CC ₅H ₁₃ ¹⁵NN ⁺, ¹³CC ₅H ₁₃N ₂ ⁺Or C ₆H ₁₃N ₂ ⁺In some embodiments, described molecular formula is selected from ¹³C ₆H ₁₃ ¹⁵N ₂ ⁺, ¹³C ₄C ₂H ₁₃ ¹⁵N ₂ ⁺With ¹³C ₃C ₃H ₁₃ ¹⁵N ₂ ⁺

Described fragment ions can produce in the following manner: make to comprise at least two kinds of not a part of ionizations of the sample mixture of the analyte molecule of isolabeling in mass spectrometer, and select at least two kinds of not analyte molecules of isolabeling with selected fracture m/z value.Then can be by applying the rupture analyte molecule of selected not isolabeling of the energy level that dissociates, the analyte molecule of wherein at least a described not isolabeling be the compound that is selected from following formula: 80 ", 81 ", 82 ", 83 ", 84 ", 85 ", 86 " and 87 ".

III. the method for mark and analysis

According to embodiments more of the present invention, the analyte mark also can be measured then.Can measure analyte, analyte self, one or more fragments of analyte and/or the fragment of label of mark by quality analysis.In some embodiments, method of the present invention can be used for the multiple analysis of the same and/or different analytes in the analysis of different analytes in the same sample and the two or more different sample.Can be with described two or more different sample mix to form sample mixture.In multiple analysis, the sample mixture that serviceable indicia reagent determination and analysis thing is originated.Can measure the absolute of the analyte that forms in two or more samples that sample mixture made up among each and/or (with respect to the same analyte in the different samples) amount (often showing with concentration or scale) is in addition relatively, but the quality analysis of operational analysis thing fragment (being sub-fragment ions) comes identification of analytes and/or analyte precursor, such as the described precursor molecule that is degraded into described analyte.

The sample that uses in the analysis can be any sample that comprises the analyte of serviceable indicia reagent mark.For example, sample can be cytolysis thing, body fluid, tissue extract or a cell extract thick or that handle.Sample can be the part from detachment process.Other possible sample type was described in this article.

Analyte in the sample can be any analyte of serviceable indicia reagent mark.For example, described analyte can be peptide and/or protein.Other possible types of analytes was described in this article.

A difference of described method is can carry out the not fact of isolabeling (i.e. coding) with unique label (it is for isomerism and/or isobaric (having identical gross mass) and differentiate the sample that described analyte is originated) from the analyte of different samples.The analyte of described not isolabeling is not distinguished under mass spectrometric MS pattern, because they have identical (always) mass-to-charge ratio.Usually the selected marker reagent set in case the analyte of mark can not distinguish by isolation technics (such as chromatography or electrophoresis) (it can be applied to potpourri before the one-level mass spectrophotometry).Yet when applying the energy level that dissociates (such as bring out dissociate (CID) by collision), described label can be formed the report thing ion of the uniqueness that can tell by the quality in the mass spectrometer (mass-to-charge ratio) by fracture.Can the relative quantity of labelled analyte in relative quantity and the sample mixture of report thing ion of detected every kind of uniqueness in the MS/MS mass spectrum and the relative quantity of the analyte in the sample of described analyte source be associated by implication (implication).Therefore, the relative intensity of report ion (being the signal ion) can be used for measuring the relative quantity of one or more analytes in the two or more different samples that are combined to form sample mixture.If the calibration standard thing of every kind of analyte that absolute quantitation is required is all introduced in the sample mixture with known quantity, then can go out the absolute magnitude (often showing) of one or more analytes in two or more samples by the information inference of report thing ion with concentration or scale.

For example, described analyte may be to utilize the enzymic digestion of handling sample to react the peptide that is formed by protein degradation.Protein degradation can be realized by utilizing one or more proteolytic enzymes (for example trypsase, papain, pepsin, ArgC, LysC, V8 proteinase, AspN, pronase, chymotrypsin or carboxypeptidase) to handle sample.Evaluation and amount by peptide in the working sample potpourri and identify the sample that it is originated, and optional other peptide of measuring this sample can be identified and/or and with respect to its sample of originating and the quantitative propeptide albumen of degrading.Because the method allows to carry out the multiple assay of protein, so under the situation more than a sample (promptly from sample mixture), it is a kind of multiple method.

Therefore, in some embodiments, the present invention relates to comprise and make the different labelled reagents reactions in two or more samples (each sample comprises one or more reactivity analytes) and the labelled reagent group and produce two or more not methods of the sample of isolabeling (every kind comprises one or more labelled analytes).Described labelled reagent can be selected from isomerism and/or isobaric labelled reagent, the report thing part of wherein said different each self-contained unique qualities of labelled reagent.Described report thing part can be any report thing part.For example, described report thing part can comprise replacement or unsubstituted piperidines, piperazine or morpholine group.

For example, the different labelled reagents in described group can comprise its salt form or hydrate forms by formula I ' representative:

Wherein atom or group Y, J, K, L, R ₁, R ₂, X ₁And X ₂As the front define and wherein said group in different labelled reagents have identical gross mass, but the group Y-J of report thing part that wherein forms each different labelled reagent at one or more isotope enrichments position by unique code, thereby when the key between the remainder of the group J of group Y-J and described labelled reagent fragment ruptures, produce report thing part in mass spectrometer with unique qualities.Atom or group Z ' can be the leaving groups of reaction active groups or reaction active groups.

For example, described reaction active groups can be N-hydroxy-succinamide ester (NHS), N-hydroxysulphosuccinimide ester (NHSS), pentafluorophenyl group ester (Pfp), 2-nitrobenzophenone ester, 3-nitrobenzophenone ester (3-NP), 4-nitrobenzophenone ester (4-NP), 2,4-dinitrophenyl ester, pentafluorophenyl group ester (Pfp), five chlorophenyl ester (Pcp), 3-hydroxyl-1,2,3-phentriazine-4 (3H)-ketone ester (Dhbt), hydroxyl pyrrolidine ketone ester (NHP), 2,4-dihalogenated phenyl ester (referring to the argumentation under Fig. 8 and the title " illustrative methods of preparation labelled reagent "), 2,2,2-trifluoroethyl ester or 1,1,1,3,3,3-hexafluoro-2-propyl diester (leaving group that is reaction active groups can be among the compound 7-19).

Therefore, the analyte of the mark of sample mixture can be by formula I " representative, comprise its salt form and/or hydrate forms:

Wherein atom or group Y, J, K, L, R ₁, R ₂, X ₁And X ₂Define as the front.For example, variable Y can be to replace or unsubstituted morpholine, piperidines or piperazine group.Group Z " can be covalently bound analyte.

Described labeling method can produce two or more not samples of isolabeling, and every kind of sample comprises the analyte of one or more marks.In case utilize the unique labelled reagent of sample is carried out mark to the analyte of every kind of sample, then can the sample of isolabeling or its be not partially mixed to produce sample mixture with two or more.Described sample mixture can randomly comprise one or more calibration standard things.

Can write down the volume and/or the amount that produce every kind of sample of sample mixture through combination.Every kind of sample is with respect to gross sample volume and/or the volume of amount and/or amount can be used to measure the certified analyte in every kind of sample in sample mixture is analyzed the amount (often showing with concentration or scale) of sample mixture.Therefore, sample mixture can comprise complex mixture, wherein can be by relative quantification to the amount of analyte in each of two or more samples, perhaps identify and/or the relative quantity of quantitatively identical and/or different analytes by the absolute quantitation that wherein in sample mixture, adds the calibration standard thing.

For example, potpourri can be applied to spectral technique, wherein can utilize the one-level mass spectrometer that described sample mixture or its part are carried out the one-level mass spectrophotometry.Can select ion then from the specific mass-to-charge ratio of described one-level mass spectrophotometry.Can apply the energy level that dissociates (being collision induced dissociation (CID)) to selected ion and cause the fracture of selected ion.Apply the energy level that dissociates by the selected ion to labelled analyte, key RL in selected ion at least a portion and LA (referring to the argumentation under the title " RL key and LA key ") can rupture.The fracture of key RL and LA can cause the fracture of report thing/connector part and cause Ionized report thing part (promptly reporting thing part or signal ion) to discharge from described analyte.Also can produce the sub-fragment ions of analyte to the fracture of selected ion by dissociation energy.Then can be with described ion (remaining selected ion, sub-fragment ions and Ionized report thing part) guiding second order ms analyser.

In described second order ms analyser, can carry out the second order ms analysis to selected ion and fragment thereof.Described second order ms analysis can be measured some or all quality (gross mass and/or absolute mass) of the sub-fragment ions of at least a labelled analyte in the gross mass (or m/z) of exist with selected mass-to-charge ratio every kind unique report thing ion and relative quantity and the sample mixture.For the every kind of analyte that exists with selected mass-to-charge ratio, can utilize described sub-fragment ions to identify a kind of and/or multiple analytes that exists with selected mass-to-charge ratio.For example, can described in the part of " by the analyte determination of computer-Aided database analysis ", carry out this analysis according to the front title.

In some embodiments, some step of described method can repeat one or many.For example, in some embodiments, as previously mentioned, can apply the energy level that dissociates to ion, thereby produce the sub-fragment ions of Ionized report thing part (promptly reporting the thing ion) and at least some selected ions from the selected mass-to-charge ratio of one-level mass spectrophotometry (different) with any previous selected mass-to-charge ratio.Can carry out the second order ms analysis to selected ion, report thing ion and sub-fragment ions or its part.Also can measure the gross mass of the report thing ion of every kind of uniqueness in the described second order ms analysis and the quality (gross mass and/or absolute mass) of relative quantity and described sub-fragment ions.In the method, can obtain can be used for from the evaluation of one or more other analytes of described one-level mass spectrophotometry and/or quantitative information.

In some embodiments, sample mixture by the classification situation of (for example separating) by chromatography or electrophoresis under, can repeat described method one or many.For example, repeat described method, can analyze whole sample mixtures by one or more other parts to sample.Can estimate, in some embodiments, but the whole process repeated one or many, and as mentioned above, in these repeat each time in, some step all can repeat one or many.In the method, can farthest seek the also content of working sample potpourri.Also can repeat described whole process to new group of two or more samples.

The those of ordinary skill of field of mass spectrometry will be seen that, can carry out the firsts and seconds mass spectrophotometry in tandem mass spectrometer.The instrument of quality analysis of being suitable for connecting was described the front in this article.Though preferred tandem mass spectrometer also can use the single-stage mass spectrometer.For example, can cause the analyte fracture, use the quadrupole mass spectrometer of single-stage or time of-flight mass spectrometer that the gained fragment is analyzed then by taper hole voltage break method.In other example, can utilize lasing light emitter that analyte is applied dissociation energy, and after post-source decay, in flight time or time-of-flight mass spectrometry instrument (TOF-TOF), write down the fragment of gained.

In some embodiments, the inventive method also can be included in before the mark that carries out sample analytes, utilizes each sample of at least a enzymic digestion with the sample component of partly or entirely degrading (in addition referring to the above-mentioned part of title for " sample preparation ").For example, described enzyme can be proteinase (with degrade proteins and/or peptide) or nuclease (with degraded nucleic acid).Can use two or more enzymes with further degraded sample component together.For example, described enzyme can be a proteolytic enzyme, such as trypsase, papain, pepsin, ArgC, LysC, V8 proteinase, AspN, pronase, chymotrypsin and carboxypeptidase (for example Carboxypeptidase A, B, C etc.).

In some embodiments, method also can be included in and carry out one-level mass spectrophotometry sample separation potpourri (in addition referring to the above-mentioned part of title for " comprising the separation of sample separation potpourri ") before.In such a way, can only carry out the one-level mass spectrophotometry to the part of sample mixture.Can pass through any separation method, comprise by chromatography and/or electrophoresis and separating.For example, can before quality analysis, use chromatogram/mass spectroscopy (LC/MS) to realize such sample separation.In addition, also can use any chromatography separating method that is suitable for separating interested analyte.The suitable chromatogram and the non-limiting example of electrophoresis separating method have been described herein.

In some embodiments, can utilize digestion and separating step to put into practice described method.Although these steps are chosen wantonly, they usually carry out together, for example, and when carrying out Proteomic analysis when being in harmonious proportion downward modulation to measure going up of intracellular protein.In some embodiments, can repeat step (having or do not have digestion and/or the separating step) one or many of described method with the analyte in evaluation and/or the quantitative sample or one or more analytes of every kind of sample in two or more samples (comprising sample) with carrier-bound labelled reagent mark.Depend in sample mixture, whether there is calibration standard, concrete sample quantitatively can be with respect to the analyte of other mark, perhaps it can be absolute.

As previously mentioned, the analysis of the quality (gross mass or absolute mass) by the antithetical phrase fragment ions can be measured the analyte relevant with selected ion.A kind of such assay method is described in the part of title for " analyte determination of analyzing by computer-Aided database ".In case determination and analysis thing then provides the basis of determining relevant sample compound out of Memory about the gross mass of the report thing ion of each uniqueness in the second order ms analysis and the information of the quality of relative quantity and sub-fragment ions.

Can determine the relative quantity of report thing ion by the peak intensity in the mass spectrogram.In some embodiments, can utilize mass spectrometer to pass through the analysis of the peak height of the report thing ion (signal ion) that obtained or peak width (or peak area) is determined the amount of the report thing ion of every kind of uniqueness.Because available different every kind of sample of labelled reagent mark, and every kind of labelled reagent can comprise the uniqueness report thing part that produces unique report thing ion, described report thing ion can be associated with the sample of the specific not isolabeling that is used to prepare sample mixture, different report thing ion determination in the second order ms analysis be can be used for identifying the sample of the not isolabeling that the report thing ion of selected analyte is originated.Under the situation of finding a plurality of report thing ions (promptly according to multiple method of the present invention), can report the relative quantity that the thing ion is determined every kind of unique report thing ion with respect to other.Because the relative quantity of measure in the second order ms analysis every kind unique report thing ion can be associated with the relative quantity of analyte in the sample mixture, so can measure every kind of not relative quantity (being often expressed as concentration and/or amount) of the analyte in the sample of the formation sample mixture of isolabeling.In addition, the quantitative information that can make analyte is associated with component in the initial not isolabeling sample, wherein the analyte of Ce Dinging is the accessory substance (be that analyte is a catabolite, such as be under the situation of the peptide that forms of the digestion by protein at analyte) of interested another kind of compound.

As mentioned above, can repeat this at the selected ion of different mass-to-charge ratioes and analyze one or many, thereby obtain to be combined to form the relative quantity of one or more other analytes in every kind of sample of sample mixture.In addition, as being described in the part of " relative quantification of analyte and absolute quantitation " at aforementioned title, in appropriate circumstances, can proofread and correct the isotopic abundance of natural or artificial generation at the peak intensity relevant with every kind of unique report thing ion.

In some embodiments, described analyte can be the peptide in sample or the sample mixture.But the analysis of the peptide in sample or the sample mixture be can be used for determining the amount (being often expressed as concentration and/or amount) of identification of protein in sample or the sample mixture, and wherein the protein in one or more samples can be degraded before the one-level mass spectrophotometry.And, the information from different samples can be compared to make mensuration, such as the effect of the amount that is used for comparison pair cell protein, described cell is hatched with influence cell growth, growth, differentiation and/or the dead material of variable concentrations.In addition, the example of indefiniteness can comprise the comparison to the expressed protein component of ill or healthy tissue or cell culture.This can be included in and utilize infectious agent (as bacterium or virus) infection or other morbid state (as cancer) back to compare expressed protein level in the cell.In other example, can carry out protein concentration (time-process) research of changing in time, with the effect of the expressed protein component of check drug therapy pair cell or tissue.In other examples, can utilize fetch from the information of different samples with detect and monitoring as tissue, organ or the biofluid of disease (for example cancer) or result of infection in the concentration of concrete protein.These experiments can comprise one or more control samples.In some embodiments, this experiment can be used for measuring interested above-mentioned two or more character.

In some embodiments, described analyte can be the nucleic acid fragment in sample or the sample mixture.The information of related nucleic acid fragment can be used to determine can identify in sample or the sample mixture amount (being often expressed as concentration and/or amount) of nucleic acid molecules, and wherein said sample was degraded before the one-level mass spectrophotometry.In addition, can be relatively from the information of different samples to make interested mensuration on the science.

The calibration standard thing that comprises unique report thing part with join the situation that the analyte that has selected mass-to-charge ratio in the sample mixture is connected with known quantity (being often expressed as concentration and/or amount) under, can utilize the absolute magnitude (being often expressed as concentration and/or amount) of analyte in every kind of sample that the described uniqueness relevant with the calibration standard thing report that the quantitative determination of thing is combined to form sample mixture.This is possible, because the amount of the analyte that is associated with the uniqueness report thing ion of correction reference material in the sample mixture is known, so can measure relative quantities of all unique report thing ions of the labelled analyte that is associated with selected ion.Since the relative quantity of every kind of unique report thing ion being surveyed of every kind of described unique report thing part (the report thing part that comprises the calibration standard thing) is not with the amount of the analyte that is associated of the sample of isolabeling is not proportional with being combined to form every kind of sample mixture, then can be based on the absolute magnitude (being often expressed as concentration and/or amount) of measuring every kind of sample analyte with respect to the calculating ratio of the preparation that is used for preparing sample mixture.When needing, can carry out the correction of the isotopic abundance of natural or artificial generation at the peak intensity that is associated with uniqueness report thing ion.Such analytical approach especially can be used for having in the Proteomic analysis of various product of complicated character, carries out before carrying out the one-level mass spectrophotometry when special under the situation of pre-separation (for example liquid chromatography or electrophoretic separation) of labelled analyte.

For example, if sample mixture comprises the calibration standard thing of 100fmol/mL, and the relative intensity of the uniqueness that is associated with described calibration standard thing report thing ion is 1, and the relative intensity of first other the unique report thing ion that is associated with first sample is 1/2nd (1/2 or 0.5), the relative intensity of second other the unique report thing ion that is associated with second sample is 2, then described first not in the isolabeling sample (mix and form sample mixture) amount (sample 1 of supposition equivalent and sample 2 mix to form sample mixture) of analyte be 50fmol/mL (0.5 x 100fmol/mL), and described second not in the isolabeling sample (mixing and the formation sample mixture) amount of analyte be 200fmol/mL (2 x 100fmol/mL).And if for example described analyte is the peptide that is associated with specified protein, then the amount of protein is 50fmol/mL in the deducibility sample 1, and the amount of protein is 200fmol/mL in the sample 2.Therefore, the existence of calibration standard thing makes and can mix and form every kind of the sample mixture not absolute quantitation of labelled analyte in the isolabeling sample (and its precursor) in some cases.

As previously mentioned, this analysis can repeat one or many at the selected ion with different mass-to-charge ratioes, thereby obtains the absolute magnitude of combination with one or more other analytes in the every kind of sample that forms sample mixture.In addition, as previously mentioned, when needed, can carry out the correction of the isotopic abundance of natural or artificial generation at the peak intensity that is associated with uniqueness report thing ion.

In some embodiments, the described herein method of labelled reagent practice of available support combination, wherein every kind of different labelled reagent is carrier-bound and is connected on the described carrier by disconnecting connector in the group, thus make every kind of different sample with described group in have different labelled reagents carrier be connected.Exemplary carrier title was in front discussed (in addition referring to Fig. 5 and the following examples part) in the part of " composition ".According to described method, can be randomly wash vehicle is reacted with the reaction active groups of removing not with labelled reagent after making the reaction of analyte and carrier-bound labelled reagent but before biased sample sample component.Form the analyte of mark and randomly carried out washing step in case made the reaction of described analyte and labelled reagent, then can discharge the analyte of mark from carrier by under the condition that disconnects the connector that can disconnect, handling carrier.In case disconnect, then can randomly gather two or more not each in the isolabeling sample, every kind of sample comprises the analyte of one or more marks, and wherein the uniqueness report thing of the analyte of the mark that is associated with specific sample by being connected thereto partly is appraisable and/or can be quantitative.Disconnect product no matter whether gather separately, all they can be mixed to form sample mixture.

In some embodiments, can utilize labelled reagent, comprise its salt form and/or hydrate, put into practice described method herein by formula II ' representative:

Wherein W, M, J, K, L, R ₁, R ₂, X ₁, X ₂And Z ' as defined above.

In some embodiments, can utilize by formula III ' labelled reagent of representative, comprise its salt form and/or hydrate, put into practice described method herein:

Wherein s, t, R ₁, R ₂, R ₁₁And Z ' as defined above.

In some embodiments, can utilize at least a labelled reagent, comprise its salt form and/or hydrate, put into practice described method herein by following various representative:

Or

R wherein ₁, R ₂And Z ' as defined above.Symbol * represents in due course ¹³C substitutes ¹²The situation of C or ¹⁵N substitutes ¹⁴The situation of N.

In some embodiments, can utilize labelled reagent, comprise its salt form and/or hydrate, put into practice described method herein by formula IV ' representative:

Wherein t, R ₁₁And Z ' as defined above.

In some embodiments, can utilize at least a labelled reagent, comprise its salt form and/or hydrate, put into practice described method herein by following various representative:

Or

Wherein ^*And Z ' as previously mentioned.

IV. proteomics workflow

In some embodiments, can before carrying out the sample preparation step, carry out the mark of sample analytes.In some embodiments, can after carrying out the sample preparation step, carry out the mark of sample analytes.In some embodiments, can between other sample preparation step, carry out the mark of sample analytes.In some embodiments, the mark of analyte be the final step of sample preparation and/or just the preparation sample mixture before.

With the example of proteome analysis as indefiniteness, several at least possible flow processs that existence may be used.For helping to understand following the argumentation, between precursor protein and analyte peptides, make differentiation sometimes.Yet, should be appreciated that in different embodiments any one or two kinds of in protein and/or the peptide all can be counted as described analyte herein.

In a kind of flow process, described precursor protein can be digested the peptide analysis thing that can be labeled subsequently.In another kind of flow process, serviceable indicia reagent is digested the peptide analysis thing of mark then with described precursor protein mark.In another flow process, described precursor protein can be captured on the solid carrier, digestion, then can be with described carrier-bound peptide-labeled.Randomly, described flow process by peptide also can be labeled.In another kind of flow process, described precursor protein can be captured on the solid carrier, mark, then can be with described carrier-bound proteopepsis to produce the peptide of mark.Randomly, described flow process by peptide also can be analyzed.No matter flow process is how, can be before MS and MS/MS analyze as required the peptide to mark carry out other sample preparation (for example separating step).

A) relate to the digestion and the exemplary flow of mark subsequently

With reference to Figure 13, for example may there be " contrast " sample to be analyzed and " test " sample.If for the example shown in Figure 13, purpose is to analyze the peptide (as analyte) of " contrast " and " test " sample protein, in some embodiments, the protein of sample can randomly be reduced, randomly halfcystine be sealed and by enzymic digestion, can be labeled the analyte peptides that is used for sequential analysis thereby produce.In some embodiments, analyte peptides is labeled (tagging) without further sample preparation.Mark howsoever, the different labelled reagents (for example labelled reagent group of isomerism and/or isobaric label) of all available each self-contained unique qualities report thing part are with the analyte mark of every kind of different samples.

In some embodiments, may carry out further sample preparation before mark and/or after the mark.For example, can carry out separating step removing the peptide of uninterested some kind, thereby reduce the complicacy of sample.Can be with the sample mix of mark to obtain sample mixture.In some embodiments, can be before mass spectrophotometry the analyte peptides of mark be separated (for example high performance liquid chromatography (HPLC)) or other classification process.

Another kind of exemplary flow is shown among Figure 14 a and Figure 15.The main different Figure 15 of being of Figure 15 and Figure 14 a for example understand the sealing that may relate to the halfcystine mercapto that peptide catches and the optional step of regeneration.For clarity sake, though Figure 13,14a, 15 and 16-18 in description all shown application at the flow process of two kinds of samples, but self-evident, as long as the different labels that can obtain other also can be handled other sample with every kind of different sample or the sample fraction of encoding.

Figure 14 a has illustrated in some embodiments and can how " contrast " sample and " test " sample have been captured sample component on the solid phase by the connector that can disconnect then with enzymic digestion.For example, carrier can comprise the connector that can disconnect and with the reaction active groups of the structure division reaction of peptide (referring to Figure 14 b illustrating) to the basis of such carrier.In Figure 14 c illustrated be suitable for catching the instantiation of the carrier of the peptide that comprises halfcystine.Comprise halfcystine mercapto peptide can with the iodoacetic acid radical reaction of illustrational carrier.Because be not that all peptides all estimate to contain halfcystine,, will do not flow through carrier and be not hunted down because do not contain those peptides of halfcystine part so this is the method that is used to reduce sample complicacy to be analyzed.In case be fixed, according to the disposal route as shown in Figure 14 a, serviceable indicia reagent with the amine groups mark of described peptide (referring to Figure 14 a).For example, every kind " contrast " sample carries out mark with different labels in all available isomerism of " test " sample and/or the isobaric labelled reagent group.The peptide analysis thing of mark can be disconnected and/or further handles (comprise and form potpourri) and/or analyze that (Figure 14 a) from carrier then.

In some embodiments, the peptide (because they are not with functional group reactions of described carrier) that flows through carrier can be collected (rather than discarding), with the labelled reagent mark in isomerism and/or the isobaric labelled reagent group and independent or analyze with the peptide of the mark of collecting from described carrier.This flow process is shown in Figure 16 and 17.As shown in Figure 16 and 17, the peptide that available identical or different isomerism and/or the labelled reagent marked flows in the isobaric labelled reagent group are crossed solid carrier.No matter labelled reagent how, all can randomly mix it with the sample mixture of analyzing by MS/MS.Also can analyze it independently.Figure 16 and 17 difference be when peptide is still on carrier (Figure 16) or with peptide after described carrier disconnects (Figure 17) but mark is retained in the peptide on the carrier.

With reference to Figure 18, can use solid carrier to catch precursor protein.Two samples that can have as exemplified, parallel processing.A kind of suitable carrier that is used for the halfcystine part of capture protein is shown in Figure 14 c.(for example flowing through) removed and randomly collected to the protein that can utilize washing will not contain the halfcystine part from carrier.Also can be randomly analyze or separate analysis with their digestion, mark and/or with sample mixture.

According to Figure 18, can be with carrier-bound protein digestibility.Can utilize labelled reagent carrier-boundly to comprise the peptide-labeled of halfcystine and disconnect (this selection is shown in Figure 18) then from carrier with described.Perhaps at first the described carrier-bound peptide that comprises halfcystine can be disconnected from carrier, use labelled reagent mark (this selection is not shown among Figure 18) then.Mark peptide (randomly comprising the mark peptide that does not contain the halfcystine part) from different samples can be mixed, handles and/or analyzes or separate analysis with sample mixture.

According to Figure 18,, can collect any peptide that discharges from carrier as the result who digests.Usually these are the peptides that do not comprise mercapto.Can randomly these peptides also randomly be mixed, handle and/or analyze or separate analysis with sample mixture with the labelled reagent mark.

B) exemplary flow that relates to mark and digest subsequently

No matter whether use carrier to catch the analyte that is used to analyze, all can before or after digestion or other chemical treatment, utilize the step of labelled reagent labelled analyte, as long as described processing does not change described label.For protein example, also reduction of sample protein matter and halfcystine can be sealed, used the side chain amido of labelled reagent mark sample protein N-ε-lysine, then protein digestibility is become the peptide of mark.

Regardless of how originating, can with the analyte analyzation of mark or further handle (comprising the preparation sample mixture), for example, by separation and/or by being fixed on the carrier.For example, can be by the pendant amine radical reaction that makes labelled reagent and sample protein N-ε-lysine labelled precursor albumen, randomly the precursor protein of described mark is fixed on the carrier then.The albumen of mark can be disconnected digestion then from described carrier, perhaps can the albumen of described mark still with during carrier combines with its digestion.Under a kind of situation in back, carrier-bound digestion will discharge the peptide that does not contain the halfcystine part from carrier.It can be collected and separate analysis or analyze randomly as a part that comprises the sample mixture that contains halfcystine mark peptide partly that the latter discharges.

When labelled precursor albumen before precursor protein is digested to peptide, can make a change digestion method.For example, utilize tryptic digestion to expect and produce the terminal arginine peptide of dominant C-, because N-is ε-the lysine side-chain amido is labeled thing and modified.Therefore, tryptic activity resembles Arg-C very much.Because only be that the terminal arginine peptide of C-that also contains the arginine side chain can be labeled and therefore can detect in mass spectrometer, so this provides the method for a kind of further minimizing sample complicacy with further processing and/or analysis.

In some embodiments, can the halfcystine disjunction mark be remembered with the protein reduction and with labelled reagent (being the specific labelled reagent of mercaptan), then the peptide of the mark that protein digestibility is become to be used to analyze.The peptide analysis thing of described mark can be analyzed or further handled, for example by separating and/or being fixed on the carrier.For example, can the peptide of mark be fixed on the carrier by the reaction between the N-α-amido on the lysine side-chain and/or N-ε-amido and carrier functional group.The carrier that can disconnect connector that has that is used for fixing the compound that comprises amidine functional group comprises that the carrier that contains the trityl connector is (referring to can be available from Novabiochem (San Diego, trityl chlorine carrier (Trityl-Cl) CA) or 2-chlorine trityl chlorine carrier).This flow process and aforementioned those are completely different.The analyte of mark can be disconnected from carrier, further handle and/or analyze.The method may not reduce complicacy in fact, because the peptide of all digestion all estimates to comprise at least one N-α-amido.

The foregoing description is not to be intended to get rid of multiple possible flow process.They only are exemplary.About the embodiment of mark before digestion, also can before digesting, implement further sample preparation.

C) sum up

Although detailed argumentation has been carried out to proteome analysis with as the protein of analyte and/or the mensuration of peptide, the multiple analytes that need too much not test but described notion is intended to comprise the application of aforementioned flow process by the mode of instantiation in the front.Therefore, the scope of the disclosure of invention is not intended to and is limited in the described instantiation of any of these.

IV. potpourri

In some embodiments, the present invention relates to potpourri (being sample mixture).For example, described potpourri can comprise with amount dystopy and/or isomeric labelled analyte.The exemplary mixture of labelled analyte and preparation thereof and/or analytical approach were described in the part of " method of mark and analysis " at aforementioned title.

Can mix forming potpourri by all or part of product with two or more labeled reactants, wherein with the different labelled reagent marks of every kind of sample with the labelled reagent group, wherein every kind of labelled reagent comprises the report thing part of uniqueness (always) quality.Can be obtained the labeled reactant in (i.e. source) by in the analyte of two or more marks each and be identified the uniqueness report thing part of every kind of different labelled reagents.Described labelled reagent can be isotope-coded isomerism and/or isobaric labelled reagent.Therefore, the analyte of two or more marks of potpourri can make isomeric and/or isobaric.The analyte of the character of labelled reagent and the mark relevant with those methods was discussed in front.

The analyte of potpourri can be a peptide.The analyte of potpourri can be a protein.The analyte of potpourri can be peptide and protein.The analyte of potpourri can be a nucleic acid molecules.The analyte of potpourri can be a carbohydrates.The analyte of potpourri can be a lipid.The analyte of potpourri can be a steroids.The analyte of potpourri can be that quality is less than 1500 daltonian micromolecule.The analyte of potpourri comprises that two or more are dissimilar (i.e. (1) lipid and steroids; Or (2) peptide, lipid, steroids and carbohydrates).

Potpourri can comprise the analyte of the not isolabeling of any kind, and described analyte comprises new report thing/connector part disclosed herein.For example, described potpourri can comprise at least two kinds not isolabeling can be by formula I " analyte of representative, comprise its salt form and/or hydrate forms:

Wherein atom or group Y, J, K, L, R ₁, R ₂, X ₁And X ₂Defined in front, its character disclosed.In some embodiments, every kind in the analyte of two kinds of marks all can be derived from different samples.According to formula I "; group Y-J (forming the not report thing part of the analyte of isolabeling) can be at one or more isotope enrichments position by unique code; thus when the key between the remainder of the analyte of the group J of group Y-J and described mark ruptures, form report thing ion with unique qualities in mass spectrometer.Group Z " can be covalently bound with analyte.For every kind of different label, in the potpourri analyte of some marks can be identical, the analyte of some marks can be different.

In some embodiments, potpourri can comprise at least two kinds not isolabeling can be by formula II " analyte of representative, comprise its salt form and/or its hydrate forms:

Wherein W, M, J, K, L, R ₁, R ₂, X ₁, X ₂And Z " as previously mentioned.

In some embodiments, potpourri can comprise at least two kinds not isolabeling can be by formula III " analyte of representative, comprise its salt form and/or its hydrate forms:

Wherein s, t, R ₁, R ₂, R ₁₁And Z " as previously mentioned.

In some embodiments, potpourri can comprise at least two kinds not isolabeling can comprise its salt form and/or its hydrate forms by the analyte of following formula representative:

Or

Wherein ^*, R ₁, R ₂, and Z " as previously mentioned.

In some embodiments, potpourri can comprise at least two kinds not isolabeling can be by formula IV " analyte of representative, comprise its salt form and/or its hydrate forms:

Wherein t, R ₁₁And Z " as previously mentioned.

In some embodiments, potpourri can comprise at least two kinds not isolabeling can comprise its salt form and/or its hydrate forms by the analyte of following formula representative:

Or

Wherein, ^*And Z " as previously mentioned.

In some embodiments, the present invention relates to the potpourri of fragment ions.For example, can produce the fragment ions of potpourri by following manner: in mass spectrometer, make to comprise at least two kinds of not a part of ionization of the sample mixture of the analyte molecule of isolabeling, and select at least two kinds of analyte molecules to be used for fracture with not isolabeling of selected m/z value.Then can be by applying the analyte molecule fracture that the energy level that dissociates makes described selected not isolabeling.In some embodiments, the analyte molecule of at least a not isolabeling is the compound that is selected from following formula: as shown in Figure 26 a-26b 80 ", 81 ", 82 ", 83 ", 84 ", 85 ", 86 " and 87 ".Therefore, fragment ions can have following molecular formula: ¹³C ₆H ₁₃ ¹⁵N ₂ ⁺, ¹³C ₄C ₂H ₁₃ ¹⁵N ₂ ⁺, ¹³C ₃C ₃H ₁₃ ¹⁵N ₂ ⁺, ¹³C ₃C ₃H ₁₃ ¹⁵NN ⁺, ¹³C ₂C ₄H ₁₃ ¹⁵NN ⁺, ¹³CC ₅H ₁₃ ¹⁵NN ⁺, ¹³CC ₅H ₁₃N ₂ ⁺Or C ₆H ₁₃N ₂ ⁺In some embodiments, described molecular formula is selected from ¹³C ₆H ₁₃ ¹⁵N ₂ ⁺, ¹³C ₄C ₂H ₁₃ ¹⁵N ₂ ⁺With ¹³C ₃C ₃H ₁₃ ¹⁵N ₂ ⁺

V. kit

In some embodiments, the present invention relates to kit.Described kit can comprise labelled reagent and one or more other reagent, container, enzyme, buffering agent and/or operation instruction as described herein.Described kit can comprise other reagent, container, enzyme, buffering agent and/or operation instruction of the group of two or more labelled reagents and one or more.Two or more labelled reagents in the kit can be isomeric and/or isobaric.For example, one or more labelled reagents in the described kit can be as the disclosed following various compound (comprising the compound group) in front herein: I ', II ', III ', IV ', V ', VI ', VII ', VIII ', IX ', X ', XI ', XII ', XIII ', XV ', XVI ', XVII ', XVIII ', XIX ', XX ', XXI ', XXII ' and/or XXIII '.In some embodiments, described kit can comprise the analyte (for example as the calibration standard thing) as the disclosed following various mark in front herein: I ", II ", III ", IV ", V ", VI ", VII ", VIII ", IX ", X ", XI ", XII ", XIII ", XV ", XVI ", XVII ", XVIII ", XIX ", XX ", XXI ", XXII " and/or XXIII ".Other character of labelled reagent is open in the kit.Described kit can for example can be used in the same sample or the multiple analysis of one or more analytes in two or more different samples.

VI. the illustrative methods for preparing labelled reagent

With reference to figure 7a-7c, Figure 27 a-27c and 20-21, the general synthesis strategy that can be used for preparing with the labelled reagent of measuring the dystopy coding will be discussed as another embodiment of the invention.The method shown in these that should be appreciated that has been represented separately and can be used for preparing with a kind of in may synthetic method of the labelled reagent of amount dystopy coding multiple.It is also understood that those of ordinary skills only by conventional test and content disclosed herein, can easily adjust this method and be used to prepare other labelled reagent with similar chemical constitution.Therefore, the disclosure of this method is intended to illustrate and is not to be eliminating or the restriction that is intended to by any way.

Although provide at uncoded compound in the method shown in Fig. 7 a-7c, Figure 27 a-27c and the 20-21, but it is self-evident, the alternative uncoded compound of reagent with amount dystopy coding, thereby producing the product with amount dystopy coding, should be substantially the same on reactivity because the material of coding is compared with uncoded material.The method that provides among Fig. 7 a-7c obtains the support of following examples 1-9.These embodiment also support the method shown in Figure 20-21.The compound of the exemplary same amount dystopy coding of the preparation of method shown in can utilizing is described in this instructions and accompanying drawing (for example Fig. 9 a-9c, 10a-10c, 12a-12e, 28a-28b and 29a-29b) and claims.

With reference to figure 7a and 27a step 1 and Figure 20-21, can make to replace or unsubstituted diamines and replacement or unsubstituted dicarboxylic acid or anhydride reaction.Reaction product is the amino acid that comprises at least one amido link (or thioamides key) that described dicarboxylic acid or acid anhydrides and described diamines are coupled together.For example, described replacement or unsubstituted diamines can have the structure shown in compound among Figure 20 200, and described replacement or unsubstituted acid anhydrides can have the structure shown in compound among Figure 20 201.The amino acid product of described reaction can have the structure shown in compound among Figure 20 202.

One or two amido of described replacement or unsubstituted diamines all can comprise the alkylating group of N-, in each figure with R ₁And/or R ₂Expression (referring to Figure 27 for example a).Described diamines can be by amido blocking group such as tert-butoxycarbonyl (t-boc) or the protection of 9-fluorenyl carbonyl (Fmoc) part.Other suitable amido blocking group and using method thereof is found in ProtectiveGroups In Organic Synthesis such as Green, Third Edition, John Wiley ﹠amp; Sons, Inc.New York, 1999.Those of ordinary skills only utilize normal experiment and content disclosed herein, promptly can select and use other suitable blocking group.

If select dicarboxylic acid, then can one of carboxylic acid group be protected by protection (such as by forming the ester (for example tert-butyl ester) of large volume) as initiation material.Should be appreciated that when using in this article described carboxylic acid or acid anhydrides can comprise the sulphur atom of the oxygen atom that replaces described two carboxylic acid groups or anhydride group.

In some embodiments, dicarboxylic acid and diamines are all not protected.Usually, protection is used so that avoid forming impurity at reaction.Yet if reagent reacting forms desired compound and/or purifying is realized easily, protection is not necessary.For example, if initiation material is symmetrical, then often can remove protection from.

It is also understood that in certain embodiments, is not that all disclosed steps all need to carry out.As be shown in the examples, the step 2 (Fig. 7 a-7c and Figure 27 a-27c) of method shown in can removing from when selecting the diamines of Fmoc protection.

With reference to Figure 20, described replacement or unsubstituted diamines can comprise by the alkyl of K representative (for example compound 200), wherein K can be formula-(CK ' ₂) _n-or-((CK ' ₂) _m-X ₃-(CK ' ₂) _m) _p-shown in the group of two amidos of bridge joint, wherein n is 2～10 integer, each m is 1～5 integer independently of one another, p is 1～4 integer, each K ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₄,-OR ₄,-SR ₄,-R ₄' OR ₄Or-R ₄' SR ₄, R wherein ₄Be alkyl, thiazolinyl, alkynyl, aryl, heteroaryl or aryl alkyl.Described replacement or unsubstituted diamines also can comprise cyclic rings such as piperazine, and one or two in two amidos of wherein said diamines is that (for example compound 205, Figure 21) for theheterocyclic nitrogen atom.In some embodiments, one or more atoms of described replacement or unsubstituted diamines can be replaced by heavy atom isotope.With reference to Figure 20 and 21, by replace or amino-acid compound that the reaction of unsubstituted diamines and acid anhydrides forms respectively by 202 and 206 representatives.

Described replacement or unsubstituted dicarboxylic acid or acid anhydrides can comprise group L (for example compound 201, Figure 20), wherein L by the formula of two carbonyls of two carboxylic acid groups of bridge joint or acid anhydrides-(CL ' ₂) _q-or-((CL ' ₂) _m-X ₃-(CL ' ₂) _m) _p-representative, wherein q is 1～10 integer, each m is 1～5 integer independently of one another, p is 1～4 integer, each L ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₅,-OR ₅,-SR ₅,-R ₅' OR ₅Or-R ₅' SR ₅, R wherein ₅Be alkyl, thiazolinyl, alkynyl, aryl, heteroaryl or aryl alkyl.Described replacement or unsubstituted dicarboxylic acid or acid anhydrides can comprise cyclic rings such as cyclohexane or cyclopentane ring, and one or two in the wherein said carboxylic moiety (or carbonyl of the acid anhydrides that forms by two carboxylic acid groups) is the substituting group of described cyclic rings.In some embodiments, the one or more atoms in described replacement or unsubstituted dicarboxylic acid or the acid anhydrides can be replaced by heavy atom isotope.

As shown in Fig. 7 a step 1, N-(t-boc)-N-methyl-ethylenediamine 101 can react with succinic anhydride 102.This reaction forms product N-methyl ethylenediamine-N '-succinic acid (N-methylethylenediamine-N '-succinate) 103.For the compound shown in formula I '-XIII ' and the VI '-XXIII ', this reaction can be represented a kind of isotope-coded method that is used for the connector part for those.The initiation material that can utilize the possible diamines of compound of described open method (except that the step 2) preparation formula I '-XIII ' and VI '-XXIII ' and acid anhydrides is in Fig. 9 a-9c and 10a-10c illustrated.

With reference to the step 2-4 of figure 7a and 7b and Figure 27 a and 27b, for the preparation carrier bound fraction purpose of (compound that comprises report thing part can finally be connected with it), the amine of the amino acid product of may command step 1 and acid functional group.As shown in Fig. 7 a step 2, the t-boc amine protecting group is replaced into the Fmoc amine protecting group, thereby produces the compound 104 of Fmoc protection.Step 3 (Fig. 7 a) in, compound is fixed on the carrier via disconnecting connector by its carboxylic acid functional, thereby has formed carrier-bound compound 105.Described carrier (described connector is fixed on it) can comprise the disconnected connector of steric restriction.The multiple carrier that can disconnect connector that comprises is well-known for those of ordinary skills.The non-limiting example of the solid carrier of steric restriction comprises: trityl chlorine carrier (trityl-Cl, Novabiochem, P/N01-64-0074), 2-chlorine trityl chlorine carrier (Novabiochem, P/N 01-64-0021), DHPP (Bachem, P/N Q-1755), MBHA (Applied Biosystems P/N400377), 4-methyl trityl chlorine carrier (Novabiochem, P/N 01-64-0075), 4-methoxyl trityl chlorine carrier (Novabiochem, P/N 01-64-0076), hydroxyl-(2-chlorphenyl) methyl-PS (Novabiochem, P/N 01-64-0345), Rink acid vectors (NovabiochemP/Ns 01-64-0380,01-64-0202) with NovaSyn TGT alcohol carrier (Novabiochem, P/N 01-64-0074).As shown in FIG., can use trityl chlorine carrier (referring to embodiment 3).

As shown in Fig. 7 b step 4, can remove the blocking group of the terminal amine of carrier-bound compound 105, thereby promote the described amine of described carrier-bound product compound 106 and the compound that comprises report thing part to react.

Therefore, the amido of amino acid product and the compound that comprises report thing part are reacted to form report thing/connector combination.As shown in Fig. 7 a-7c and 27a-27c, in some embodiments, can promote this reaction on the solid carrier by described connector is fixed to.Yet, though should be appreciated that in some embodiments available, connector part fixing and nonessential.

The compound that comprises report thing part has two features usually.A feature can be to replace or the alkylating acetate part of unsubstituted N-, and carboxylic acid (or thiocarboxylic acid) base of wherein said acetate part can form amido link (or thioamides key) with the amido reaction of amino acid product.Second feature can be the nitrogen-atoms with the mesomethylene carbon covalent bonding of described acetate part.The part that comprises nitrogen-atoms can be the secondary amine that replaces, as dimethylamine, diethylamine or propylamine.The part that comprises nitrogen-atoms can be a ring compound, as replacing or unsubstituted piperidines, piperazine or morpholine.Shown in Fig. 7 b, described nitrogen atom partly is N-methyl-piperazine.

With reference to Figure 20 and 21, can make described amino acid product (being respectively 202 and 206) and comprise compound (the being compound 203) reaction of reporting thing.The product of this reaction can form report thing/connector part (promptly being respectively compound 204 and 207).

With reference to figure 7b step 5, can make terminal amine and the replacement or unsubstituted N-alkyl piperazine acetate part (107) reaction of compound 106, thereby form carrier-bound report thing/connector composition 108.

Certainly the compound that comprises report thing part can comprise one or more with amount dystopy enrichment positions.For example, alkylating acetate part of the N-of replacement or unsubstituted Fig. 7 c or N-alkyl piperazine can comprise one or more with amount dystopy enrichment positions (in addition referring to Fig. 9 a-9c and 10a-10c).Therefore, can and comprise the coding of compound (for example replacing or unsubstituted N-alkyl piperazine acetate) of report thing part and the coding of control report thing and connector according to diamines, diacid (or acid anhydrides).

In some embodiments, the carboxylic acid group of the molecule of representative report thing/connector or thiocarboxylic acid base can be by in-situ activations, thereby promote the mark of analyte.Therefore, in some embodiments, do not need extra reaction.

In some embodiments, the acidic group of the molecule of representative report thing/connector combination or sulfo-acidic group can be modified with form can with the reaction active groups of the functional group reactions of analyte.In some embodiments, this may relate to the reaction that produces the reagent of desired reaction active groups with one or more, described reaction active groups can with the functional group reactions of analyte.In some embodiments, this may relate to acid groups or the thio-acid group conversion of compounds to activation.For example, the carboxylic acid group or the thiocarboxylic acid base of described report thing/connector combination can be activated, thereby preparation comprises the active ester of alcohol or mercaptan leaving group, wherein said active ester can form the analyte of mark with the functional group reactions of analyte.

With reference to Figure 20 and 21, compound (being respectively compound 204 and 207) that can representative report thing/connector compound is modified, thereby produces respectively by I ' or I " compound of representative.For example, carboxylic acid group or thiocarboxylic acid base can be converted into active ester, such as the N-hydroxy-succinamide ester.

With reference to figure 7c step 6, described report thing/connector compound can be disconnected from carrier, thereby produce intermediate 109.As shown, described intermediate comprises the carboxylic acid group, and described carboxylic acid group can be activated the reaction that is used for nucleophilic thing (such as the amido of protein or peptide).Although in some embodiments, described carboxylic acid can with reference to figure 7c step 7, for example be understood to form N-hydroxy-succinamide ester 111 by the reaction with trifluoroacetic acid N-hydroxy-succinamide ester (110) as previously mentioned by in-situ activation.Other method that is suitable for forming the active ester of report thing/connector compound is found among the common unsettled and total U.S. publication application No.US 2005-0148771A1.Exemplary synthetic Fig. 8 that is shown in.The leaving group of some exemplary active ester is described as 7-19.

Fig. 9 a-c for example understands the initiation material of the same amount dystopy coding that may originate, described initiation material can with shown in method one be used from preparation compound V '-XIII '.Figure 10 a-c, wherein Bian Ma piperazine can be replaced by the N-methyl ethylenediamine derivant among Fig. 7 a-7c, for example understands the initiation material of the same amount dystopy coding that may originate, and described initiation material can be used for preparing compounds X V '-XXIII '.Be used for that (method such as the piperazine of derivant (for example methyl amimoacetic acid) the preparation coding of the aminoacetic acid of coding and aminoacetic acid is as known in the art from simple, the initiation material that is easy to get.For example, be used for preparing the piperazine of coding of all dissimilar (flavor) and the method for N-alkyl piperazine compound and be found in common unsettled and total U.S. publication application No.US2004-0219685 A1, US 2005-0147982, US 2005-0147985 and 2005-1048774A1.Figure 12 d and 12e also for example understand the example of the synthetic route of the diethylenediamine compound that is shown in the coding among Figure 10 c.Be shown among Figure 12 d and the 12e synthetic route based on as disclosed patented claim described in the method for diethylenediamine compound of preparation coding.

As previously mentioned, the initiation material of coding (such as aminoacetic acid, methyl amimoacetic acid and the succinic anhydride of coding) can derive from different commercial source, such as Cambridge Isotope Laboratories, Andover, MA (referring to Www.isotope.comOn tabulation or " basic initiation material ") and Isotec (branch offices of Sigma-Aldrich).Cambridge Isotope Laboratories and Isotec also prepare needed compound according to the synthetic agreement of client.The same, the source of the initiation material of the various dystopy of amount together codings and the list of references of numbering of part are found in the disclosure of invention everywhere, comprise in embodiment and the accompanying drawing.Yet these lists of references are only for providing the purpose of information, are not that to be intended to be restriction or all possible commercial source of limit to the present invention for required protection.

With reference to figure 11a, 11b and embodiment 8～9,, illustrated and described the method for the N-methyl-aminoacetic acid (being methyl amimoacetic acid) that is used to prepare various codings by being suitable for known synthetic reaction.For example, the raw material of these codings can be used for preparing the N methyl piperazine acetate part of coding, and described N methyl piperazine acetate partly is used in the report thing described in a plurality of U.S. publication application of being quoted at Fig. 9 a-c and 10a-c.Therefore, obviously these illustrate with embodiment and can be used for from the feedstock production of the commercially available simple coding labelled reagent of described various codings herein.

With reference to figure 12a, 12b, 12c, 28a and 29a,, for example understand the method for the N-methyl-ethylenediamine that is used to prepare various codings by being suitable for known synthetic reaction.Therefore, obviously these illustrate and can be used for from the feedstock production of the commercially available simple coding labelled reagent of described various codings herein.For example,, can estimate, at Michel etc. with reference to figure 12c Structural Study of bonding in thioamides.Synthesis and conformation of Thioalanines and thioglvcines.Canadian Journal of Chemistry, 67 (8): the method described in the 1312-1318 (1989) can be used as carry out shown in the general guide principle of step 1 of reaction.In addition, can estimate, by Gallery Chemical (a BASF Company, Florham Park, New Jersey, USA) the product document about borine-tetrahydrofuran complex that provides (and wherein disclosed list of references, as Amedia etc., Syn.Comm.29:2377 (1999)) can be used as the general guide principle of the step 2 of reaction shown in carrying out.

Embodiment:

Can be according to following examples, the each side of further understanding the present invention and being instructed, described embodiment should not be construed as the scope that limits this instruction by any way.

Embodiment 1: N-[2-(tert-butoxycarbonyl-methyl-amino)-ethyl]-succinic acid (103) synthetic (step 1-Fig. 7 a)

(5.5g 0.032mol) is dissolved in methylene chloride (CH to well-beaten N-(tert-butoxycarbonyl)-N-methyl-ethylenediamine 101 ₂Cl ₂, disposable adding succinic anhydride 102 in solution 30mL) (3.2g, 0.032mol).After at room temperature reaction mixture being stirred 1 hour, obtain slurry.This slurry is used for step 2 without further handling.

Annotate: if possible, use N-(9H-fluorenes-9-ylmethoxy carbonyl)-N-methyl-ethylenediamine carry out step 2.Reason for this reason, Fig. 9 a-c has shown the use of the N-methyl ethylenediamine of Fmoc protection.

Embodiment 2: N-{2-[(9H-fluorenes-9-ylmethoxy carbonyl)-methyl-amino]-ethyl }-succinic acid (104) (step 2-Fig. 7 a) for synthetic

The CH that is dissolved in that in the slurry of the compound 103 that produces by step 1, adds 20mL 80% ₂Cl ₂In trifluoroacetic acid (TFA).Then reaction mixture is at room temperature stirred, and by thin-layered chromatography (TLC) monitoring.After 2 hours, the TLC of potpourri shows the initiation material complete obiteration, and has formed the polarity product.In Rotary Evaporators, remove excessive solvent and TFA then and with residue and tetrahydrofuran (THF, 30ml x 3) coevaporation.Oily residue with remainder is dissolved in the acetone (50ml) then, and by the careful NaHCO that adds ₃(water) solution and pH is transferred to alkalescence.(Fmoc-Osu, 13.5g 0.0399mol) are dissolved in solution in the acetone (70mL) to disposable then adding N-(9-fluorenyl methoxy carbonyl-oxygen base) succinimide.Then reaction mixture is at room temperature stirred and spend the night.The TLC that carry out this moment has confirmed that initiation material exhausts.In Rotary Evaporators, remove acetone then, dilute residue, and wash (Et with ether with water (50mL) ₂O, 30mL x 3) to remove nonpolar impurity.With 2NHCl water layer is acidified to pH～2 then and with ethyl acetate extraction (EtOAc, 100mL x 3).Water (30mL x 4), salt solution (1 x 30mL) wash the organic moiety that merges then, and with sodium sulphate (Na ₂SO ₄) drying.In Rotary Evaporators, remove EtOAc then, and residue remained on spend the night under the high vacuum and obtain hydroscopicity white solid N-{2-[(9H-fluorenes-9-ylmethoxy carbonyl)-methyl-amino]-ethyl-succinic acid 104 (10.5g, 82%).MS：397.2(MH+)

Embodiment 3: N-{2-[(9H-fluorenes-9-ylmethoxy carbonyl that carrier engages)-methyl-amino]-ethyl }- (step 3-Fig. 7 a) for succinic acid (105) synthetic

To N-{2-[(9H-fluorenes-9-ylmethoxy carbonyl)-methyl-amino]-ethyl }-(595mg 1.5mmol) is dissolved in CH to succinic acid (104) ₂Cl ₂Add N in the solution (10mL), (DIPEA, 776mg 6mmol), add trityl chlorine carrier (1g, 1mmol, P/N SC5028, Advanced Chemtech) to the N-diisopropylethylamine then.At room temperature slurry was stirred 1 hour then, use CH then ₂Cl ₂Solution, the CH of/MeOH/DIPEA (17:2:1,3 x 10mL) ₂Cl ₂(3 x 10mL), N, CH is used in dinethylformamide (DMF, 2 x 10mL) washing at last again ₂Cl ₂(2 x 10mL) washing.The Fmoc of carrier of dry load (105) and analytic sample load under vacuum.Average load is 0.5mmol/g.This carrier is used for step 4 without further handling.

Embodiment 4: Synthetic (the step of N-(2-methylamino-ethyl)-succinic acid (106) that carrier engages Rapid 4-Fig. 7 b)

Piperidines/DMF with 20% (use 10mL, allow then to discharge) handles succinic acid N-(Fmoc)-N-methyl ethylenediamine (105) that carrier engages.And then add piperidines/DMF (10mL) of 20% and stirred slurry 10 minutes.Use DMF (10mL x 2), CH then ₂Cl ₂(10mL x 2) wash vehicle is used DMF (10mL x 2) washing at last.Carrier (106) with deprotection is used for step 5 rapidly then.

Embodiment 5: The N-that carrier engages (2-{ methyl-[2-(4-methyl-piperazine-1-yl)-acetyl group]-amino }- Ethyl)-synthetic (step 5-Fig. 7 b) of succinic acid (108)

With (4-methyl-piperazine-1-yl)-acetate two (trifluoroacetic acid) salt 107 (965mg, 2.5mmol) be dissolved in dry DMF (8mL) and DIPEA (646mg, 5mmol) in.In this solution, add O-(7-azepine benzo triazol-1-yl)-N, N, N ', N '-tetramethylurea hexafluorophosphate (HATU, 950mg, 2.5mmol).With potpourri vortex 1 minute and to wherein adding carrier (106).Slurry was stirred 25 minutes, filter, with DMF (10mL x 3), CH ₂Cl ₂(10mL x 2) and DMF (10mL x 2) washing.Use the reagent of equivalent that carrier is carried out secondary coupling (second round of coupling) (being double couple crosslinking (double coupling)) then, use DMF (10mL x 3) and CH then ₂Cl ₂(10mL x 3) washing.Under vacuum, be used for step 6 then with carrier (108) drying and without further handling.

Embodiment 6: N-(2-{ methyl-[2-(4-methyl-piperazine-1-yl)-acetyl group]-amino }-ethyl)-amber Synthetic (step 6-Fig. 7 c) of acid (109)

Carrier (108) is used TFA/CH ₂Cl ₂(10mL) handle and place 5 minutes.Solvent is discharged from carrier, use TFA/CH once more ₂Cl ₂(15mL) handle carrier and collect solvent.With TFA/CH ₂Cl ₂Two parts mix, in Rotary Evaporators, concentrate then.With residue with THF (20mL x 3) coevaporation.Under high vacuum, remove the TFA of trace.Grind residue with absolute ether then.Obtain gluing agglomerate.Utilizing stirring rod that this product is placed under the strong agitation spends the night.By the white solid of centrifuging gained,, dry and obtain white hydroscopicity solid 109 (320mg, overall yield 59%) under high vacuum with ether washing (5mL x 3).MS：315(MH+)

Embodiment 7: N-(2-{ methyl-[2-(4-methyl-piperazine-1-yl)-acetyl group]-amino }-ethyl)-amber Synthetic (step 7-Fig. 7 c) of the NHS ester (111) of acid

To 109 (100mg, 0.18mmol) be dissolved in add in the solution of anhydrous THF (2mL) trifluoroacetic acid N-hydroxy-succinamide ester 110 (48mg, 0.22mmol).Reaction mixture was at room temperature stirred 2 hours.Under Rotary Evaporators, remove THF, by removing the TFA of trace with extra THF (2mL x 2) coevaporation.In refrigerator, gluey residue is statically placed in the absolute ether then and spends the night.Decant goes out ether and residue is remained on to obtain foamed 111 (110mg, 93%) under the high vacuum.MS：412(MH+)

Embodiment 8: Synthesizing (a) of N-(the t-boc)-methyl amimoacetic acid (21) of coding referring to Figure 11

Anhydrous THF (100ml) is transferred to the BOC-that accommodates of purging with nitrogen gas by sleeve pipe ¹⁵NH- ¹³CH ₂- ¹³COOH (7g, 39.29mmol, 1 equivalent, Cambridge IsotopeLab is P/N:CNLM-2412-0) and in the 500mL round-bottomed flask of magnetic stirring bar.Potpourri is at room temperature stirred up to obtaining clear solutions.Utilize ice bath that solution is cooled to 0 ℃ then.Is furnished with the graduated cylinder of partition and to wherein shifting potassium tert-butoxide (t-BuOK, 157mL, 1M, in THF, 4 equivalents) solution with purging with nitrogen gas.Then described t-BuOK solution is joined and utilize sleeve pipe and pressure differential to stir down in the reaction mixture simultaneously at 0 ℃.Originally formed gel, described gel dissolved in about one minute.Make to be reflected at and stirred again under 0 ℃ 10 minutes.

With number ampoules ¹³CH ₃I (2 x 10g, 140mmol, 3.56 equivalents, CambridgeIsotope Lab, P/N:CLM-287-10) refrigerator and cooled but ( ³CH ₃I is highly volatile, should only open under freezing) and under nitrogen covers, open.Content is transferred in the reaction mixture rapidly by sleeve pipe.Then 0 ℃ of following strong agitation reaction mixture 1 hour.Little aliquot (the 100 μ L) water (1mL) of reaction is handled, is acidified to pH1 and uses EtOAc (2mL) extraction by adding 1M HCl.The TLC of EtOAc layer the analysis showed that to react and finishes (R _f20=0.51, R _f21=0.70; 1:4 MeOH-CH ₂Cl ₂-AcOH (10 μ l/10mL); Make the TLC colour developing by the solution heating of triketohydrindene hydrate in ethanol (EtOH) with 3% (w/v).

Under reduced pressure remove the THF solvent then, solid is dissolved in the 100mL water, be acidified to pH=1 with 1M HCl then.With EtOAc (300mL x 2) extraction water medium.With the EtOAc layer that merges with 2% (w/v) NaHSO ₃(50mL)+salt solution (50mL) solution-treated and mixing to remove the I that in processing procedure, forms strongly ₂The EtOAc layer is further washed and uses Na with salt solution (50mL) ₂SO ₄Dry.Under reduced pressure remove EtOAc and any tert-butyl alcohol (t-BuOH) then and obtain the compound 21 (7.42g, productive rate 97%) of stiff water white oil or low melting point solid.The MNa that ES-MS (directly adding in the methyl alcohol (MeOH)) calculates ⁺(C ₅ ¹³C ₃H ₁₅ ¹⁵NO ₄Na)=216.10, the MNa of actual measurement ⁺=216.10.

Embodiment 9: Synthetic (referring to Figure 11 b) of the methyl amimoacetic acid methyl esters (23) of coding

With BOC- ¹⁵N ¹³CH ₃- ¹³CH ₂- ¹³COOH (21,7.42g, 38.41mmol), dimethyl aminopyridine (DMAP, 470mg, 3.41mmol) and MeOH (7.8mL, 5 x 38.41mmol) be dissolved among the EtOAc (200mL) and be cooled to 0 ℃.In reaction mixture, add the dicyclohexylcarbodiimide (DCC, 8.32g, 1.05x 38.41mmol) that is dissolved among the EtOAc (30mL) then.Then reaction mixture is stirred and spend the night, be warmed to room temperature simultaneously gradually.TLC the analysis showed that reaction finishes (R _f21=0.00, R _f22=0.50; 7:3 hexane-EtOAc; Make the TLC colour developing by the solution heating of triketohydrindene hydrate in EtOH) with 3% (w/v).Remove dicyclohexylurea (DCU) (DCU) precipitation and filtrate is concentrated into 50mL by filtering (Whatman #2 filter paper).Absorb into concentrated solution in the silica gel and (carry out twice by purification by flash chromatography; 120g SiO ₂, post Isco.; 85mL/min was dissolved in 10% EtOAc of hexane in 0～5 minute, was dissolved in 25% EtOAc of hexane in 5～20 minutes).

Merge and contain the fraction of pure products 22 and use Rotary Evaporators at room temperature to be concentrated into 50mL (boiling point of compound 22 is low, should not apply high vacuum).In concentrated solution, add HCl (60mL, 4M HCl is in dioxane) and stirring.Observe strong generation bubble.After 30 minutes, TLC the analysis showed that the protection (R that sloughs t-Boc fully _f23=0.00, R _f22=0.50; 7:3 hexane-EtOAc; Make the TLC colour developing by the solution heating of triketohydrindene hydrate in EtOH) with 3% (w/v).Under reduced pressure remove volatile matter and obtain white hydroscopicity solid chemical compound 23 (5.2g, two step overall yields 94%).The 4MH that ES-MS (directly adding in the methyl alcohol (MeOH)) calculates ⁺(C ¹³C ₃H ₁₀ ¹⁵NO ₂)=108.09, the MH of actual measurement ⁺=108.08, the M of calculating ₂H ⁺=215.18, the M of actual measurement ₂H ⁺=215.17.

Embodiment 10: [the Glu that carrier engages ¹ ]-human fibrin peptide B's is synthetic

[Glu ¹]-human fibrin peptide B[Glu-Fib, CAS#:103213-49-6]: Fmoc-peptide synthetic schemes (the Novabiochem catalog that utilizes standard, 2004-2005) and following amino acid derivativges at trityl chloride resin (P/N:Novabiochem, 01-64-0074) go up artificial assembling described peptide: Fmoc-Arg (Pbf)-OH (P/N:Novabiochem, 04-12-1145), Fmoc-GIu (O ^tBu)-OH (P/N:Novabiochem, 04-12-1020), Fmoc-Gly-OH (P/N:Novabiochem, 04-12-1001), Fmoc-Val-OH (P/N:Novabiochem, 04-12-1039), Fmoc-Asn (Trt)-OH (P/N:Novabiochem, 04-12-1089), Fmoc-Asp (Mpe)-OH (P/N:Bachem, B-3560), Fmoc-Phe-OH (P/N:Novabiochem, 04-12-1030), Fmoc-Ser ( ^tBu)-OH (P/N:Novabiochem, 04-12-1033), Fmoc-Ala-OH (P/N:Novabiochem, 04-12-1006).[Glu ¹The amino acid sequence of]-human fibrin peptide B: Seq ID.No.1:Glu-Gly-Val-Asn-Asp-Asn-Glu-Glu-Gly-Phe-Phe-Ser-Ala-Arg.

Embodiment 11: Fmoc-N (Me)-CH ₂ CH ₂ -N (Me)-CO-CH ₂ CH ₂ -COOH's is synthetic

With NH (Me)-CH ₂CH ₂-NH (Me) (1.6mL, 15mmol, P/N:Alfa-Aesar, USLF006653) solution that is dissolved in THF (15mL) joins trityl chloride resin (P/N:Novabiochem, 01-64-0074,1g, 1.5mmol) in and stirred suspension at ambient temperature 1 hour.With resin filter and with N-N-methyl-2-2-pyrrolidone N-(NMP, 15mL x 3) washing, handled 15 minutes then with MeOH-DMF-diisopropylethylamine (DIPEA, 15mL, 4:7:2 v/v, P/N:Aldrich, 387649).Wash with resin filter and with NMP (15mL x 3) at last.

In resin, add then the succinic anhydride be dissolved among the DMF (10mL) (P/N:Aldrich, 2399690,1.5g, 15mmol), add subsequently DIPEA (2.61mL, 15mmol).Agitating resin 20 minutes at ambient temperature then.Then with resin filter and with NMP (15mL x 3) washing, then with acetonitrile (CH ₃CN, 15 x 3mL) washing.

(20% v/v 40mL) handles, filters and use once more TFA-DCM (20% v/v, 10mL x 5) washing with TFA-DCM with resin.Under reduced pressure filtrate is condensed into oily residue (TFA, N (Me)-CH ₂CH ₂-N (Me)-CO-CH ₂CH ₂-COOH 1.07g), is dissolved in saturated NaHCO with described residue ₃In (pH 8-9).The solution (4.24mmol is dissolved in the acetone (20mL) for P/N:Advance ChemTech RC8015,1.43g) that adds Fmoc-OSu then in described aqueous solution also stirred 2 hours at ambient temperature.TLC the analysis showed that and formed product (R _f=0.50; 9:1:0.01 DCM-MeOH-AcOH, UV 254nm makes the TLC colour developing by the solution heating of triketohydrindene hydrate in EtOH with 3% (w/v)).Concentrated reaction mixture is to remove acetone, then with water (150mL) dilution residue then.By utilizing Et ₂Non polar impurities is removed in O (100mL x 2) extraction.(pH～1, HCl 1M) and with EtOAc (100mL x 2) extract with the water layer acidifying.The EtOAc layer that merges is passed through Na ₂SO ₄Dry and concentrate and obtain the colourless toughening oil Fmoc-N of 0.95g (Me)-CH ₂CH ₂-N (Me)-CO-CH ₂CH ₂-COOH.The MH that ES-MS (directly adding among the MeOH) calculates ⁺(C ₂₂H ₂₄N ₂O ₅H ⁺)=397.17, the MH of actual measurement ⁺397.16.

Embodiment 12: Fmoc-NH-CH ₂ CH ₂ -NH-CO-CH ₂ CH ₂ -COOHDIPEA's is synthetic

Make Fmoc-NH-CH ₂CH ₂-NH ₂HCl (P/N:Novabiochem, 01-63-0064,1 equivalent (eqv.)) reacts in DCM with succinic anhydride (1eqv.) in the presence of DIPEA (1eqv.) and obtains described title compound.

Embodiment 13: Fmoc-NH-CH ₂ CH ₂ -N (Me)-CO-CH ₂ CH ₂ -COOH's is synthetic

To well-beaten Boc-NMe-CH ₂CH ₂-NH ₂(265mg 1.52mmol) is dissolved in the solution that adds Fmoc-OSu (564mg, 1.67mmol are dissolved in the 15mL acetone) in the solution of acetone (15mL).Stirred the mixture at ambient temperature then 2 hours.In this stage the TLC of reaction mixture be the analysis showed that and to have formed Boc-NMe-CH ₂CH ₂-NH-Fmoc (R _f=0.35; 3:7 EtOAc: hexane, UV 254nm makes the TLC colour developing by the solution heating of triketohydrindene hydrate in EtOH with 3% (w/v)).

Behind the evaporation acetone with product by flash chromatography (254nm, 3:7 EtOAc: hexane, collect the 18mL fraction, fraction 15-24 contains pure product for 40g Isco-silica column, 40mL/min) purifying, obtain foamed Boc-NMe-CH ₂CH ₂-NH-Fmoc (520mg, productive rate=86%).

Use TFA-water (15ml, 95:5, v/v) treatments B oc-NMe-CH at ambient temperature ₂CH ₂(520mg, 1.31mmol) 1 hour, this moment, TLC the analysis showed that the protection that has removed Boc fully to-NH-Fmoc.Under reduced pressure remove TFA-water and the oil of gained is dissolved among the DCM (30mL).(131mg 1.31mmol), adds DIPEA (to pH～10 (by wet pH test paper)) then to add succinic anhydride in this solution.Stirred the mixture then 30 minutes.Extract with reaction mixture HCl (1M) acidifying (pH=1) and with EtOAc (100mL x 3) then.Wash the EtOAc layer that merges and pass through Na with salt solution (100mL x 2) ₂SO ₄Dry.Under reduced pressure remove EtOAc and obtain the title compound of colorless oil.The MH that ES-MS (directly adding among the MeOH) calculates ⁺(C ₂₃H ₂₆N ₂O ₅H ⁺)=411.10, the MH of actual measurement ⁺411.09.

Embodiment 14: Synthetic (Figure 20) of N-(Fmoc)-N '-succinyl group-piperazine

To the Boc-piperazine (P/N:Lancaster L13363,500mg, 2.68mmol) be dissolved in add in the solution of DCM (30ml) succinic anhydride (269mg, 2.68mmol).Stirring reaction is 2 hours at ambient temperature.TLC the analysis showed that the Boc-piperazine (R that has formed succinylation _f=0.50; 9:1:0.01 DCM-MeOH-AcOH makes the TLC colour developing by the solution heating of triketohydrindene hydrate in EtOH with 3% (w/v)).In this solution, add TFA (30mL), stirred the mixture at ambient temperature then 1 hour.Under reduced pressure remove the volatile ingredient in the potpourri and the oil of gained is dissolved among the THF (30mL) that contains minimal amount of water, by adding DIPEA pH regulator to 9.(907mg, 2.69mmol) solution that is dissolved among the THF (10mL) also stirred 1 hour at ambient temperature to add Fmoc-OSu.TLC the analysis showed that and formed product (R _f=0.55; 9:1:0.01 DCM-MeOH-AcOH, UV 254nm makes the TLC colour developing by the solution heating of triketohydrindene hydrate in EtOH with 3% (w/v)).Under reduced pressure remove the volatile ingredient in the potpourri then and the oil of gained is dissolved in the saturated NaHCO of minimum volume ₃In.Use Et then ₂O (100mL x 2) extraction water solution extracts with HCl (1M) acidifying (pH～1) and with EtOAc (150mL x 2) again.The EtOAc layer that merges is passed through Na ₂SO ₄Dry and concentrate and obtain the colorless oil product.

Embodiment 15: The coupling of the Fmoc-diamines of succinylation and piperazine acetate and Glu-Fib peptide:

[the Glu that the carrier of about 10mg is engaged ¹]-human fibrin peptide B resin (referring to embodiment 10) is handled (2mL x 1 minute filters, and 2mL x is 5 minutes then) with the piperidines that 20% (v/v) is dissolved among the DMF, filters and washing (NMP).With the Fmoc-diamines of succinylation (or its DIPEA salt, as shown in the embodiment 11-14 [except being used to prepare compound 125, but the Fmoc-NH-CH that can utilize disclosed herein method make homemade from irrelevant raw material ₂CH ₂CH ₂CH ₂-NH-CO-CH ₂CH ₂Beyond-the COOH], with respect to the 10eqv of Glu-Fib amount on the resin) with HATU (P/N:Applied Biosystems 4317033,9.5eqv) and DIPEA (30eqv) NMP (～activate in 1mL).Join the compound of described activation in the resin and mixed 30 minutes.With resin filter, wash then, and remove the Fmoc group by the piperidines processing that aforesaid usefulness 20% (v/v) is dissolved among the DMF with NMP.Utilize then HATU (9.5eqv) and DIPEA (60eqv) NMP (～1.5mL) in the activation piperazine acetate-tfa salt (10eqv).Solution with the compound of this activation joins in the resin then.After 30 minutes resin is washed with NMP, use CH then ₃The CN washing.Use 95:5 TFA-water (200 μ L, 2 hours) that the peptide of mark is disconnected (and deprotection) and uses ether (Et from resin then ₂O) precipitation.

The analytical data of mass spectrum of various mark peptides (ES-MS, directly input in water)

Compound 120:(N-methyl-piperazine) acetyl group-N (Me)-CH ₂CH ₂-N (Me)-CO-CH ₂CH ₂-CO-Glu-Fib: the MH of calculating ⁺=1880.9, the MH of actual measurement ⁺=1880.3

Compound 121:(N-methyl-piperazine) acetyl group-NH-CH ₂CH ₂-NH-CO-CH ₂CH ₂-CO-Glu-Fib: the MH of calculating ⁺=1853.8, the MH of actual measurement ⁺=1853.8

Compound 122:(N-methyl-piperazine) acetyl group-NH-CH ₂CH ₂-N (Me)-CO-CH ₂CH ₂-CO-Glu-Fib: the MH of calculating ⁺=1867.9, the MH of actual measurement ⁺=1867.9

Compound 123:(N-methyl-piperazine) acetyl group-N (Me)-CH ₂CH ₂-NH-CO-CH ₂CH ₂-CO-Glu-Fib: the MH of calculating ⁺=1867.9, the MH of actual measurement ⁺=1867.9

Compound 124:(N-methyl-piperazine) acetyl group-piperazine-CO-CH ₂CH ₂-CO-Glu-Fib: the MH of calculating ⁺=1879.9, the MH of actual measurement ⁺=1879.0

Compound 125:(N-methyl-piperazine) acetyl group-NH-CH ₂CH ₂CH ₂CH ₂-NH-CO-CH ₂CH ₂-CO-Glu-Fib: the MH of calculating ⁺=1880.90, the MH of actual measurement ⁺=1880.94.

Embodiment 16: Discussion about Figure 22 a, 22b, 23a, 23b, 24a and 24b

Figure 22 a is the MS analysis chart of compound 122.Observe the mark peptide to have+2 and the strong peak of the ion of+3 electric charges.Figure 22 b be to selected observed in Figure 22 a+the MS/MS analysis chart of 2 peaks and its fragment.Except the sub-fragment ions of peptide, also observe strong signal at the uncoded report thing ion at m/z 113.10 places.These data show that the labelled reagent of the coding of same general structure will rupture and produces the report thing ion with unique qualities, and described report thing ion can be used for the multiple analysis of analyte.

Figure 23 a is the MS analysis chart of compound 124.The peptide of observing mark has+and 2 and the strong peak of the ion of+3 electric charges.Figure 23 b be to selected observed in Figure 23 a+the MS/MS analysis chart of 2 peaks and its fragment.Except the sub-fragment ions of peptide, also observe strong signal at the uncoded report thing ion at m/z113.10 place.These data show that the labelled reagent of the coding of same general structure will rupture and produces the report thing ion with unique qualities, and described report thing ion can be used for the multiple analysis of analyte.

Figure 24 a is the MS analysis chart of compound 125.The peptide of observing mark has+and 2 and the peak of the ion of+3 electric charges.Figure 24 b be to selected observed in Figure 24 a+the MS/MS analysis chart of 2 peaks and its fragment.Except the sub-fragment ions of peptide, also observe strong signal at the uncoded report thing ion at m/z 113.10 places.These data show that the labelled reagent of the coding of same general structure will rupture and produces the report thing ion with unique qualities, and described report thing ion can be used for the multiple analysis of analyte.

Under MS and MS/MS analysis, compound 120,121 and 123 also shows and compound 122,124 and 125 viewed similar quality.Under all scenario, report thing ion and sub-fragment ions have all been observed.Therefore, these data show that the labelled reagent of the coding of same general structure will rupture and produces the report thing ion with unique qualities, and described report thing ion can be used for the multiple analysis of analyte.

Embodiment 17: the N-of coding (t-boc)-ethylaminoethanol synthetic (Figure 28 a)

(Figure 28 a) for step 1

Utilize sleeve pipe under Ar Pressure to N-Boc-methyl amimoacetic acid (C ₃, ¹⁵N) 140(10g 51.7mmol) is dissolved in the BH that drips 1M in the ice-cold solution of anhydrous THF (200ml) ₃THF be dissolved in solution among the THF (15.56g, 181ml, 181mmol).Originally control adds the effervesce that speed keeps appropriateness.In case effervesce finishes, just increase adding speed.After the adding, stirred the mixture about 1.5 hours at 0 ℃.Finish by the reaction of TLC analysis confirmation.To react quencher by the methyl alcohol of 0 ℃ of careful adding then.Concentrated reaction mixture in Rotary Evaporators.Then with oily residue and methyl alcohol (500mL) coevaporation that adds in addition.Utilize the Combi-flash instrument, with the oily residue of gained by purification by flash chromatography obtain 8g (90%) colorless oil the N-Boc-N-Me-ethylaminoethanol ( ¹³C, ¹⁵N) 141MS(178，M+H)

Step 2, (Figure 28 a):

Surpass under 4 minutes time to 141 (7.8g, 48mmol), Et ₃(16.7mL 120mmol) is dissolved in and adds MsCl in the ice-cold solution of DCM (700mL) (P/N:Fluka 64260, and 4.5mL 57.6mmol) and simultaneously stirs N.After spending 15 minutes again under 0 ℃,, show to have formed new product (R by the TLC analyze reaction mixture _f2=0.20, R _f3=0.42; 1:1 hexane-EtOAc makes the TLC colour developing by the solution heating of triketohydrindene hydrate in EtOH with 3% (w/v)).

Evaporation DCM also is dissolved in yellow solid among the EtOAc (1.5L).(salt solution (400mL x 2) washing is used in 1M, 800mL) washing then, passes through Na with HCl with the EtOAc layer ₂SO ₄Dry and concentrate and obtain yellow oil.Described oil is passed through column chromatography (120g SiO ₂Post Isco (X2); 40mL/ minute, be dissolved in the 35%EtOAc of hexane in 0～5 minute, be dissolved in the 40%EtOAc of hexane in 5～15 minutes.Collect a plurality of 18mL-fractions; Fraction 9-24 contains pure compound) purifying and obtain the Boc of colorless oil ^*NH ^*CH ₂CH ₂OMs 142 (～11.5g), it is directly used in next step.

Step 3; Figure 28 a

With the THF of minimum (～60ml) with compound 142 (48mmol, suppose that the productive rate that reacts previously is 100%) transfer in the chemical glass pressure vessel, and to the methylamine solution that wherein adds 500mL2M (P/N:Aldrich 395056,31g, 1000mmol), seal and (cover safe in utilization) spends the night in 40～45 ℃ of down heating (stirring simultaneously).TLC analyzes (1:1 EtOAc-hexane) and shows that 142 exhaust fully and generated new product (R _f4=0.52; 1:1DCM-MeOH+1% (v/v) Et ₃N; Earlier the TLC plate is heated 5 minutes to remove Et ₃N makes its colour developing by the solution heating of triketohydrindene hydrate in EtOH with 3% (w/v) then).Then reaction mixture is concentrated in Rotary Evaporators, be dissolved in residue in the methylene chloride (1.5L) and transfer in the separating funnel.Add 1N NaOH (200mL) and salt solution (200mL) and extraction.Further use 1N NaOH (200mL) and salt solution (200mL) washing organic layer.Pass through Na ₂SO ₄Dry.Residue is obtained 143 (6.45g, 76%) of water white oil by Combi Flash purifying.MS(177M+H)。

Embodiment 18: the Fmoc-NH-CH that comprises 6 isotope-coded positions ₂ CH ₂ -N (Me)-CO- CH ₂ CH ₂ Synthetic (Figure 28 b) of-COOH:

To stir 143 (6.4g, 36.5mmol) be dissolved in disposable adding succinic anhydride in the solution of acetone (200mL)- ¹³C ₄(3.8g.36.5mmol).(5mL 36.5mmol) and with reaction mixture at room temperature stirred 30 minutes to drip triethylamine then.TLC confirms initiation material complete obiteration at this moment.Concentrated reaction mixture and residue is dissolved in the methylene chloride (100mL) in Rotary Evaporators.The HCl that adds 140mL 4M then is dissolved in the solution in the dioxane.In 5 minutes, form white precipitate.By TLC analytically clear liquid to confirm the complete obiteration of initiation material.Concentrated reaction mixture and the white solid residue is dissolved in saturated NaHCO in Rotary Evaporators ₃In the solution (pH 8-9).(14.5g, 43mmol) solution that is dissolved in the acetone (400mL) also at room temperature stirred 3 hours to add Fmoc-Osu then in above-mentioned solution.At this moment TLC show the reaction finish.Concentrated reaction mixture in Rotary Evaporators, and use ether (200mL) then and water (200mL) are transferred to solid residue in the separating funnel.Tell ether layer, and with ether (200mL X 3) aqueous layer extracted.With 6M HCl diethyl ether solution is acidified to pH 2 and uses EtOAc (4 X 200mL) extraction then.The EtOAc extract that merges washed with salt solution (50mL X 3) and pass through Na ₂SO ₄Dry.On Rotary Evaporators, evaporate EtOAc, obtain foam, this foam is remained on obtain white solid 144, productive rate 95% under the high vacuum.MS(403，M＝H)

Embodiment 19: (Figure 29 a) for the N-of coding (Fmoc)-N '-(methyl)-ethylenediamine HCl synthetic

Step 1 (Figure 29 a):

Utilize sleeve pipe under Ar Pressure to the N-Boc-methyl amimoacetic acid ( ¹³C ₃, ¹⁵N) 150(10g 51.7mmol) is dissolved in the BH that drips 1M in the ice-cold solution of anhydrous THF (200ml) ₃THF be dissolved in solution among the THF (15.56g, 181ml, 181mmol).Originally control adds the effervesce that speed keeps appropriateness.In case effervesce finishes, just increase adding speed.After the adding, stirred the mixture about 1.5 hours at 0 ℃.Finish by the reaction of TLC analysis confirmation.To react quencher by the methyl alcohol of 0 ℃ of careful adding then.Concentrated reaction mixture in Rotary Evaporators.Then with oily residue and methyl alcohol (500mL) coevaporation that adds in addition.Utilize the Combi-flash instrument, with the oily residue of gained by purification by flash chromatography obtain 8g (90%) colorless oil the N-Boc-N-Me-ethylaminoethanol ( ¹³C ₃, ¹⁵N) 151MS(180，M+H)

Step 2 (Figure 29 a):

Utilize sleeve pipe under Ar Pressure to the N-Boc-N-Me-ethylaminoethanol ( ¹³C ₃, ¹⁵N) 151(8g, 44.6mmol), carbon tetrabromide (17.8g, 53.5mmol) and sodium azide (8.7g adds dry DMF (240ml) in potpourri 133.8mmol), and stirs the slurry 5 minutes of gained.(14g 53.5mmol) is dissolved in the solution in the anhydrous dimethyl formamide (45ml) and preserving under argon gas to prepare triphenylphosphine in independent flask.Utilize sleeve pipe under Ar Pressure, in above-mentioned slurry, to drip described triphenylphosphine solution.Use DMF (5ml) flushing flask to guarantee quantitative transfer again.Warm reaction mixture shows glassy yellow.At room temperature stirred the mixture then 30 minutes.Take out aliquot and add several ether.Be settled out white solid, by TLC clear liquid analytically.Shown finishing of reaction by the disappearance of initial alcohol (Rf:0.24) and the formation of less polarity product (Rf:0.65).In reaction mixture, add ether (1.5L) with precipitation triphenyl phosphonium oxide.Filter the slurry of gained then by the sinter funnel that diatomite layer is arranged.Use ether (500ml) fully to wash white solid once more.Use the filtrate of salt solution (300ml X 4) washing merging then and pass through Na ₂SO ₄Drying is filtered and is evaporated on Rotary Evaporators under the vacuum tightness of appropriateness being lower than under 25 ℃ the temperature.Use the Combi-Fash instrument residue to be obtained the azide of water white oil by purification by flash chromatography then 152, it is used for next step rapidly without any analysis.

Step 3 (Figure 29 a):

The azide that will obtain from above-mentioned steps 152Be dissolved in the methyl alcohol (900ml).Add saturated ammonium chloride solution (180ml) then and reaction mixture was at room temperature stirred 5 minutes.Then to reaction mixture small batch ground add zinc powder (8.7g, 133.8mmol).Having observed nitrogen emits.Stirred reaction mixture 10 minutes at room temperature then.Analyze aliquot by TLC, confirm finishing of reaction by the disappearance of initiation material (Rf:0.65).Leach solid (unreacted zinc and zinc hydroxide) and use methyl alcohol (200ml) washing.In Rotary Evaporators, concentrate the filtrate that merges.Residue is dissolved in the methylene chloride also with 6N NaOH solution (800mL) extraction.With methylene chloride (800ml) back extraction water layer.The dichloromethane extract that merges is passed through Na ₂SO ₄Dry and evaporation and obtain the Boc-amine 153 of light yellow stiff oil in Rotary Evaporators.Step 2 and 3 overall yield are 80%.MS(179，M+H)

Step 4 (Figure 29 a):

To N-Me-Boc-amine 153(5.62g adds Fmoc-Osu (10.64g, acetone 31.5mmol) (200ml) solution in acetone 31.5mmol) (200ml) solution.At room temperature stirred reaction mixture added the saturated NaHCO of 80mL after 5 minutes ₃Solution.Continued stirred reaction mixture 2 hours.TLC shows the initiation material complete obiteration and has formed the product of less polarity at this moment.In Rotary Evaporators, evaporate acetone.Semi-solid residue is distributed in EtOAc (2L), 1M HCl (125ml), salt solution (125mL) and water (250mL).The EtOAc layer is further used 1M HCl (100mL), salt solution+water (150mL+250mL) and salt solution (250ml X 2) washing.Pass through Na ₂SO ₄Dry and evaporation and obtain linen solid product (12.6g) in Rotary Evaporators is dissolved in it in methylene chloride (200mL) and handled 30 minutes and obtain the Fmoc amine hydrochlorate of white solid with 4N HCl (188mL) 154(10.4g).MS(301，M+H)

Embodiment 20: The Fmoc-NH-CH that comprises 8 isotope-coded positions ₂ CH ₂ -N (Me)-CO- CH ₂ CH ₂ Synthetic (Figure 29 b) of-COOH

To the Fmoc-amine hydrochlorate 154(7.25g, disposable adding succinic anhydride in methylene chloride 21.5mmol) (600mL) suspension ( ¹³C ₄).Drip Et then ₃N (4.5ml, 32.3mmol).At room temperature stirred reaction mixture is after 30 minutes, and TLC the analysis showed that initiation material complete obiteration.In Rotary Evaporators, evaporate methylene chloride.The residue of gained is distributed in EtOAc (2L) and 1M HCl (350mL).Organic layer is further used 1M HCl (200mL) and salt solution (200mL X 4) washing, pass through Na ₂SO ₄Drying is also evaporated and is obtained white foam, and described white foam was remained on high vacuum following 48 hours, obtains the Fmoc-NH-CH of the coding of white solid ₂CH ₂-N (Me)-CO-CH ₂CH ₂-COOH 155 (7.5g, 86%).MS(405，M+1)

Utilize basically as the synthetic schemes (in addition referring to Fig. 7 a-7c) described in the above-mentioned embodiment 1-7, the compositions according to embodiment 19 and 20 preparations can be used for synthetic according to the labelled reagent of the coding of Figure 27 a-27c.Figure 25 a-25b for example understands some labelled reagents that can utilize disclosed conventional method preparation among the present invention.In addition, Figure 26 a-26b for example understands the analyte that can utilize some marks of disclosed labelled reagent preparation among Figure 25 a-25c.According to the fracture in mass spectrometer, the analyte of described mark can be used for the fragment ions of the analyte in the quantitative above-mentioned various samples and the potpourri of fragment ions with generation.

It is also understood that by selecting the initiation material of suitable coding said method combines with the normal experiment method and can be used for preparing various other the compositions of coding, described composition can be used as the labelled reagent according to one or more inventions disclosed herein.Therefore, the foregoing description is not restrictive under any way.

Although described instruction of the present invention, be not to be intended in these embodiments with instruction restriction of the present invention together with multiple embodiments.On the contrary, it will be understood to those of skill in the art that instruction of the present invention comprise variously substitute, modification and equivalent.

Claims (72)

1. the compound of formula I representative comprises its salt form and/or hydrate forms:

Wherein,

Y can be substituted or unsubstituted and can randomly can be disconnected to be connected to 5,6 or 7 yuan of heterocycles on the carrier, and wherein said heterocycle comprises by covalent bond and is connected at least one theheterocyclic nitrogen atom on the group J;

J is by formula-CJ ' ₂The group of-representative, wherein each J ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₃,-OR ₃,-SR ₃,-R ₃' OR ₃Or-R ₃' SR ₃

K be by formula-(CK ' ₂) _n-or-((CK ' ₂) _m-X ₃-(CK ' ₂) _m) _pThe group of-representative, wherein n is 2～10 integer, each m is 1～5 integer independently of one another, p is 1～4 integer, each K ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₄,-OR ₄,-SR ₄,-R ₄' OR ₄Or-R ₄' SR ₄

L be by formula-(CL ' ₂) _q-or-((CL ' ₂) _m-X ₃-(CL ' ₂) _m) _pThe group of-representative, wherein q is 1～10 integer, each m is 1～5 integer independently of one another, p is 1～4 integer, each L ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₅,-OR ₅,-SR ₅,-R ₅' OR ₅Or-R ₅' SR ₅

Perhaps

1) R ₁Be hydrogen, deuterium or R ₆, and R ₂Be hydrogen, deuterium or R ₇

Perhaps

2) R ₁And R ₂Be together by formula-(CR ' ₂) _qOr-((CR ' ₂) _m-X ₃-(CR ' ₂) _m) _pThe group of-representative, described group have formed the ring of two nitrogen-atoms of bridge joint, and wherein q is 1～10 integer, and each m is 1～5 integer independently of one another, and p is 1～4 integer, each R ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₆,-OR ₆,-SR ₆,-R ₆' OR ₆Or-R ₆' SR ₆

X ₁Be=O ,=S ,=NH or=NR ₇

Each X ₂Be independently of one another=O or=S;

Each X ₃Be independently of one another-O-or-S-; With

Z is-OH ,-SH ,-O ^-V ⁺,-S ^-V ⁺, reaction active groups, the leaving group of reaction active groups or the analyte of covalent bonding;

Wherein,

V ⁺It is positively charged gegenion;

Each R ₃, R ₄, R ₅, R ₆And/or R ₇Be alkyl, thiazolinyl, alkynyl, aryl, heteroaryl or aryl alkyl independently of one another; With

Each R ₃', R ₄', R ₅' and/or R ₆' can be alkylidene, alkenylene, alkynylene, arlydene or alkylidene aryl independently of one another.

2. the compound of claim 1 wherein forms report thing group Y-J partly and comprises at least one isotope enrichment position.

3. the compound of claim 1, the group by formula I# representative that wherein forms the counterbalance part comprises at least one isotope enrichment position:

Wherein,

K be by formula-(CK ' ₂) _n-or-((CK ' ₂) _m-X ₃-(CK ' ₂) _m) _pThe group of-representative, wherein n is 2～10 integer, each m is 1～5 integer independently of one another, p is 1～4 integer, each K ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₄,-OR ₄,-SR ₄,-R ₄' OR ₄Or-R ₄' SR ₄

L be by formula-(CL ' ₂) _q-or-((CL ' ₂) _m-X ₃-(CL ' ₂) _m) _pThe group of-representative, wherein q is 1～10 integer, each m is 1～5 integer independently of one another, p is 1～4 integer, each L ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₅,-OR ₅,-SR ₅,-R ₅' OR ₅Or-R ₅' SR ₅

Perhaps

1) R ₁Be hydrogen, deuterium or R ₆, and R ₂Be hydrogen, deuterium or R ₇

Perhaps

2) R ₁And R ₂Be together by formula-(CR ' ₂) _q-or-((CR ' ₂) _m-X ₃-(CR ' ₂) _m) _pThe group of-representative, described group have formed the ring of two nitrogen-atoms of bridge joint, and wherein q is 1～10 integer, and each m is 1～5 integer independently of one another, and p is 1～4 integer, each R ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₆,-OR ₆,-SR ₆,-R ₆' OR ₆Or-R ₆' SR ₆

X ₁Be=O ,=S ,=NH or=NR ₇

Each X ₂Be independently of one another=O or=S; With

Each X ₃Be independently of one another-O-or-S-.

4. the compound of claim 1 wherein forms the group by formula Y-J representative of reporting the thing part and comprises at least one isotope enrichment position, and the group by formula I# representative of formation counterbalance part comprises at least one isotope enrichment position:

Wherein,

Y can be substituted or unsubstituted and can randomly can be disconnected to be connected to 5,6 or 7 yuan of heterocycles on the carrier, and wherein said heterocycle comprises by covalent bond and is connected at least one theheterocyclic nitrogen atom on the group J;

J is by formula-CJ ' ₂The group of-representative, wherein each J ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₃,-OR ₃,-SR ₃,-R ₃' OR ₃Or-R ₃' SR ₃

K be by formula-(CK ' ₂) _n-or-((CK ' ₂) _m-X ₃-(CK ' ₂) _m) _pThe group of-representative, wherein n is 2～10 integer, each m is 1～5 integer independently of one another, p is 1～4 integer, each K ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₄,-OR ₄,-SR ₄,-R ₄' OR ₄Or-R ₄' SR ₄

L be by formula-(CL ' ₂) _q-or-((CL ' ₂) _m-X ₃-(CL ' ₂) _m) _pThe group of-representative, wherein q is 1～10 integer, each m is 1～5 integer independently of one another, p is 1～4 integer, each L ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₅,-OR ₅,-SR ₅,-R ₅' OR ₅Or-R ₅' SR ₅

Perhaps

1) R ₁Be hydrogen, deuterium or R ₆, and R ₂Be hydrogen, deuterium or R ₇

Perhaps

2) R ₁And R ₂Be together by formula-(CR ' ₂) _q-or-((CR ' ₂) _m-X ₃-(CR ' ₂) _m) _pThe group of-representative, described group have formed the ring of two nitrogen-atoms of bridge joint, and wherein q is 1～10 integer, and each m is 1～5 integer independently of one another, and p is 1～4 integer, each R ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₆,-OR ₆,-SR ₆,-R ₆' OR ₆Or-R ₆' SR ₆

X ₁Be=O ,=S ,=NH or=NR ₇

Each X ₂Be independently of one another=O or=S; With

Each X ₃Be independently of one another-O-or-S-.

5. the compound of claim 1 by formula II representative, comprises its salt form and/or hydrate forms:

Wherein,

W is atom or the group that replaces at least one M group of hexa-member heterocycle, and is positioned at ortho position, a position or the contraposition of the nitrogen of described hexatomic ring, and be-N (H)-,-N (R ")-,-N (R " ')-,-P (R ")-,-P (R " ')-,-O-or-S-;

Each remaining group M is-CM ' independently of one another ₂-, wherein each M ' can be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₈,-OR ₈,-SR ₈,-R ₈' OR ₈Or-R ₈' SR ₈

J is by formula-CJ ' ₂The group of-representative, wherein each J ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₃,-OR ₃,-SR ₃,-R ₃' OR ₃Or-R ₃' SR ₃

K be by formula-(CK ' ₂) _n-or-((CK ' ₂) _m-X ₃-(CK ' ₂) _m) _pThe group of-representative, wherein n is 2～10 integer, each m is 1～5 integer independently of one another, p is 1～4 integer, each K ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₄,-OR ₄,-SR ₄,-R ₄' OR ₄Or-R ₄' SR ₄

L be by formula-(CL ' ₂) _q-or-((CL ' ₂) _m-X ₃-(CL ' ₂) _m) _pThe group of-representative, wherein q is 1～10 integer, each m is 1～5 integer independently of one another, p is 1～4 integer, each L ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₅,-OR ₅,-SR ₅,-R ₅' OR ₅Or-R ₅' SR ₅

Perhaps 1) R ₁Be hydrogen, deuterium or R ₆, and R ₂Be hydrogen, deuterium or R ₇

Perhaps

2) R ₁And R ₂Be together by formula-(CR ' ₂) _q-or-((CR ' ₂) _m-X ₃-(CR ' ₂) _m) _pThe group of-representative, described group have formed the ring of two nitrogen-atoms of bridge joint, and wherein q is 1～10 integer, and each m is 1～5 integer independently of one another, and p is 1～4 integer, each R ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₆,-OR ₆,-SR ₆,-R ₆' OR ₆Or-R ₆' SR ₆

X ₁Be=O ,=S ,=NH or=NR ₇

Each X ₂Be independently of one another=O or=S;

Each X ₃Be independently of one another-O-or-S-;

Each R " can be alkyl, thiazolinyl, alkynyl, aryl, heteroaryl or aryl alkyl independently of one another;

Each R " ' be H ₂N-R ₉'-, H (R ₁₀) N-R ₉'-, (R ₁₀) ₂N-R ₉'-, HO-R ₉'-, HS-R ₉'-or described compound be can be disconnected to the disconnected connector that couples together with carrier;

Z is-OH ,-SH ,-O ^-V ⁺,-S ^-V ⁺, reaction active groups, the leaving group of reaction active groups or the analyte of covalent bonding;

Wherein,

V ⁺It is positively charged gegenion;

Each R ₃, R ₄, R ₅, R ₆, R ₇, R ₈And/or R ₁₀Be alkyl, thiazolinyl, alkynyl, aryl, heteroaryl or aryl alkyl independently of one another; With

Each R ₃', R ₄', R ₅', R ₆', R ₈' and/or R ₉' can be alkylidene, alkenylene, alkynylene, arlydene or alkylidene aryl independently of one another.

6. the compound of claim 5 by the formula III representative, comprises its salt form and/or hydrate forms:

Wherein,

S is 1～5 integer, and t is 1～10 integer;

R ₁Be hydrogen, deuterium or R ₆

R ₂Be hydrogen, deuterium or R ₇

R ₁₁Be hydrogen, deuterium, methyl ,-C (H) ₂D ,-C (H) D ₂,-CD ₃, other alkyl or-R " ';

Z is-OH ,-SH ,-O ^-V ⁺,-S ^-V ⁺, reaction active groups, the leaving group of reaction active groups or the analyte of covalent bonding;

Wherein,

V ⁺It is positively charged gegenion;

R " ' be H ₂N-R ₉'-, H (R ₁₀) N-R ₉'-, (R ₁₀) ₂N-R ₉'-, HO-R ₉'-, HS-R ₉'-or described compound be can be disconnected to the disconnected connector that couples together with carrier; With

Wherein,

Each R ₆, R ₇And/or R ₁₀Be alkyl, thiazolinyl, alkynyl, aryl, heteroaryl or aryl alkyl independently of one another; With

Each R ₉' be alkylidene, alkenylene, alkynylene, arlydene or alkylidene aryl independently of one another.

7. the compound of claim 6, wherein said compound comprises its salt form and/or hydrate forms by following various representative:

Or

Wherein,

^*Expression comprises alternative in due course ¹²C's ¹³C and alternative ¹⁴N's ¹⁵The isotope enrichment position of N;

R ₁Be hydrogen or R ₆

R ₂Be hydrogen or R ₇

Z is-OH ,-SH ,-O ^-V ⁺,-S ^-V ⁺, reaction active groups, the leaving group of reaction active groups or the analyte of covalent bonding;

Wherein,

V ⁺It is positively charged gegenion; With

R ₆And/or R ₇Be alkyl, thiazolinyl, alkynyl, aryl, heteroaryl or aryl alkyl independently of one another.

8. the compound of claim 6, wherein said compound comprises its salt form and/or hydrate forms by following various representative:

9. the compound of claim 5 by formula IV representative, comprises its salt form and/or hydrate forms:

Wherein,

T is 1～10 integer;

R ₁₁Be hydrogen, deuterium, methyl ,-C (H) ₂D ,-C (H) D ₂,-CD ₃Or-R " ';

Z is-OH ,-SH ,-O ^-V ⁺,-S ^-V ⁺, reaction active groups, the leaving group of reaction active groups or the analyte of covalent bonding;

Wherein,

V ⁺It is positively charged gegenion;

R " ' be H ₂N-R ₉'-, H (R ₁₀) N-R ₉'-, (R ₁₀) ₂N-R ₉'-, HO-R ₉'-, HS-R ₉'-or described compound be can be disconnected to the disconnected connector that couples together with carrier; With

Wherein,

Each R ₉' be alkylidene, alkenylene, alkynylene, arlydene or alkylidene aryl independently of one another; With

Each R ₁₀' be alkyl, thiazolinyl, alkynyl, aryl, heteroaryl or aryl alkyl independently of one another.

10. the compound of claim 9, wherein said compound comprises its salt form and/or hydrate forms by following various representative:

Or

Wherein,

^*Expression comprises alternative in due course ¹²C's ¹³C and alternative ¹⁴N's ¹⁵The isotope enrichment position of N; With

Z is-OH ,-SH ,-O ^-V ⁺,-S ^-V ⁺, reaction active groups, the leaving group of reaction active groups or the analyte of covalent bonding;

Wherein, V ⁺It is positively charged gegenion.

11. each compound in the claim 1,5,6 or 9, wherein said compound comprises at least one isotope enrichment position.

12. each compound in the claim 1,5,6 or 9, wherein said compound comprises two or more isotope enrichment positions.

13. each compound among the claim 1-12, wherein Z is peptide or protein.

14. each compound among the claim 1-12, wherein Z is-OH.

15. each compound among the claim 1-12, wherein Z is the leaving group of active ester.

16. the compound of claim 15, wherein Z is a N-hydroxy-succinamide.

17. each compound among the claim 1-13, wherein said compound are the calibration standard things.

18. a method comprises:

A) make different labelled reagent reaction in every kind of two or more samples that comprise one or more reactivity analytes and the labelled reagent group, thereby form two or more not samples of isolabeling, the sample of every kind of mark comprises the analyte of one or more marks, different labelled reagent in wherein said group comprises its salt form and/or hydrate forms by formula I ' representative:

Wherein,

Y can be substituted or unsubstituted and can randomly can be disconnected to be connected to 5,6 or 7 yuan of heterocycles on the carrier, and wherein said heterocycle comprises by covalent bond and is connected at least one theheterocyclic nitrogen atom on the group J;

J is by formula-CJ ' ₂The group of-representative, wherein each J ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₃,-OR ₃,-SR ₃,-R ₃' OR ₃Or-R ₃' SR ₃

K be by formula-(CK ' ₂) _n-or-((CK ' ₂) _m-X ₃-(CK ' ₂) _m) _pThe group of-representative, wherein n is 2～10 integer, each m is 1～5 integer independently of one another, p is 1～4 integer, each K ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₄,-OR ₄,-SR ₄,-R ₄' OR ₄Or-R ₄' SR ₄

L be by formula-(CL ' ₂) _q-or-((CL ' ₂) _m-X ₃-(CL ' ₂) _m) _pThe group of-representative, wherein q is 1～10 integer, each m is 1～5 integer independently of one another, p is 1～4 integer, each L ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₅,-OR ₅,-SR ₅,-R ₅' OR ₅Or-R ₅' SR ₅

Perhaps 1) R ₁Be hydrogen, deuterium or R ₆, and R ₂Be hydrogen, deuterium or R ₇

Perhaps

2) R ₁And R ₂Be together by formula-(CR ' ₂) _q-or-((CR ' ₂) _m-X ₃-(CR ' ₂) _m) _pThe group of-representative, described group have formed the ring of two nitrogen-atoms of bridge joint, and wherein q is 1～10 integer, and each m is 1～5 integer independently of one another, and p is 1～4 integer, each R ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₆,-OR ₆,-SR ₆,-R ₆' OR ₆Or-R ₆' SR ₆

X ₁Be=O ,=S ,=NH or=NR ₇

Each X ₂Be independently of one another=O or=S;

Each X ₃Be independently of one another-O-or-S-;

Z ' is the leaving group of reaction active groups or reaction active groups;

Wherein,

Each R ₃, R ₄, R ₅, R ₆And/or R ₇Be alkyl, thiazolinyl, alkynyl, aryl, heteroaryl or aryl alkyl independently of one another;

Each R ₃', R ₄', R ₅' and/or R ₆' be alkylidene, alkenylene, alkynylene, arlydene or alkylidene aryl independently of one another; With

Wherein,

Every kind of different labelled reagent has identical gross mass in described group, but wherein the group Y-J of the formation of every kind of different labelled reagent report thing part is encoded uniquely at one or more isotope enrichments position, thereby when the key between the remainder of the group J of group Y-J and described labelled reagent ruptures, produce report thing ion in mass spectrometer with unique qualities; With

B) mix two or more not sample or its part and one or more optional calibration standard things of isolabeling, thereby produce sample mixture.

19. the method for claim 18 also comprises:

C) sample mixture or its part are carried out the one-level mass spectrophotometry;

D) apply the energy level that dissociates for ion, thereby form the report thing ion and the sub-fragment ions of at least some selected ions from the analyte of the mark with selected mass-to-charge ratio of described one-level mass spectrophotometry; And

E) selected ion, report thing ion and sub-fragment ions or its part are carried out the second order ms analysis.

20. the method for claim 19 also comprises:

F) measure the gross mass of every kind of report thing ion in the described second order ms analysis and the gross mass and/or the absolute mass of relative quantity and some or all sub-fragment ions.

21. the method for claim 19 also comprises:

G), determine the analyte of the mark relevant with selected mass-to-charge ratio by the analysis of antithetical phrase fragment ions.

22. the method for claim 21 also is included under the different selected mass-to-charge ratioes selected ion repeating step (d) to the analyte of mark to (g) one or many.

23. the method for claim 22 comprises that also repeating step (c) arrives (g) one or many, uses the different piece of sample mixture at every turn.

24. each method in the claim 18～23, wherein group Y replaces or unsubstituted morpholine, piperidines or piperazine part.

25. each method in the claim 18～24, wherein every kind of sample is thick or cytolysis thing, body fluid, tissue extract or the cell extract handled.

26. each method in the claim 18～24, wherein every kind of sample is the fraction that is obtained by detachment process.

27. each method in the claim 18～26, wherein one or more analytes are peptide and/or protein.

28. each method in the claim 18～26, wherein said reaction active groups by Z ' representative is the leaving group of active ester, described active ester is N-hydroxy-succinamide ester (NHS), N-hydroxysulphosuccinimide ester (NHSS), pentafluorophenyl group ester (Pfp), 2-nitrobenzophenone ester, 3-nitrobenzophenone ester (3-NP), 4-nitrobenzophenone ester (4-NP), 2,4-dinitrophenyl ester, pentafluorophenyl group ester (Pfp), five chlorophenyl ester (Pcp), 3-hydroxyl-1,2,3-phentriazine-4 (3H)-ketone ester (Dhbt), hydroxyl pyrrolidine ketone ester (NHP), 2,4-dihalogenated phenyl ester, 2,2,2-trifluoroethyl ester or 1,1,1,3,3,3-hexafluoro-2-propyl diester.

29. each method in the claim 19～27, wherein every kind of sample with report thing ion evaluation of unique qualities as the source of the analyte of every kind of mark.

30. each method in the claim 18～29, every kind of different labelled reagent is that carrier engages and is connected with carrier by disconnecting connector in wherein said group, thereby the carrier that makes every kind of different sample and be loaded with different labelled reagents in described group reacts; Wherein said method also comprises the following steps: before carrying out step (b)

I) randomly wash described carrier to remove not the sample component with the reaction active groups reaction of labelled reagent;

The described connector that disconnects is disconnected, thereby two or more of release confession collection are the sample of isolabeling not, every kind of sample comprises the analyte of one or more marks, and wherein the analyte of the mark that is associated with specific sample partly is appraisable by the report thing with unique qualities that is connected thereto and/or can be quantitative; With

Iii) before biased sample, randomly gather every kind of sample of the analyte of mark.

31. the method for claim 18 also comprises:

C) with the component of every kind of sample of at least a enzymic digestion with degrade partially or completely described sample or sample mixture; Or

D) separate described sample mixture; Or

E) above-mentioned c) and d).

32. the method for claim 21 wherein is determined at the every kind of report thing ion that has unique qualities in the described second order ms analysis and reports the relative quantity of thing ions with respect to other.

33. the method for claim 32, the relative quantity of wherein will the be relevant every kind report thing ion with unique qualities with certified analyte be combined to form every kind of described sample mixture not the amount of the sample of isolabeling be associated, thereby determine two or more amounts of the analyte among every kind in the sample of isolabeling or not of being combined to form described sample mixture.

34. the method for claim 33, wherein:

What i) described sample mixture comprised known quantity is used for describedly by the calibration standard thing of identification of analytes, and this calibration standard thing comprises the report thing part with unique qualities; And measure the absolute magnitude of every kind of report thing ion with reference to the amount of the uniqueness report thing ion relevant with unique qualities with described calibration standard thing; And

Ii) measure in every kind of different samples of described sample mixture by the absolute magnitude of identification of analytes with reference to relative quantity with every kind of unique qualities different report thing ion.

35. the method for claim 33, also be included in different selected mass-to-charge ratio places to the selected ion repeating step (d) of the analyte of mark to (g) one or many, thereby identify and/or be determined at two or more relative quantities of one or more other analytes among every kind of sample of isolabeling not that are combined to form described sample mixture.

36. the method for claim 34, also be included in different selected mass-to-charge ratio places to the selected ion repeating step (d) of the analyte of mark to (g) one or many, thereby identify and/or be determined at two or more absolute magnitudes of one or more other analytes among every kind of sample of isolabeling not that are combined to form described sample mixture.

37. the method for claim 34, wherein said analyte is a peptide, and based on two or more evaluation of one or more peptides among every kind of sample of isolabeling and relative quantity two or more not evaluation and the relative quantities of one or more protein among every kind of sample of isolabeling of determining to be combined to form described sample mixture not that are combined to form described sample mixture.

38. the method for claim 35, wherein said analyte is a peptide, and based on two or more evaluation of one or more peptides among every kind of sample of isolabeling and absolute magnitude two or more not evaluation and the absolute magnitudes of one or more protein among every kind of sample of isolabeling of determining to be combined to form sample mixture not that are combined to form described sample mixture.

39. the method for claim 18, wherein said labelled reagent is by formula III ' representative, comprise its salt form and/or hydrate forms:

Wherein,

S is 1～5 integer, and t is 1～10 integer;

R ₁Be hydrogen, deuterium or R ₆

R ₂Be hydrogen, deuterium or R ₇

R ₁₁Be hydrogen, deuterium, methyl ,-C (H) ₂D ,-C (H) D ₂,-CD ₃, other alkyl or-R " ';

Z ' is the leaving group of reaction active groups or reaction active groups;

Wherein,

R " ' be H ₂N-R ₉'-, H (R ₁₀) N-R ₉'-, (R ₁₀) ₂N-R ₉'-, HO-R ₉'-, HS-R ₉'-or described compound be can be disconnected to the disconnected connector that couples together with carrier; With

Wherein,

Each R ₆, R ₇And/or R ₁₀Be alkyl, thiazolinyl, alkynyl, aryl, heteroaryl or aryl alkyl independently of one another; With

Each R ₉' be alkylidene, alkenylene, alkynylene, arlydene or alkylidene aryl independently of one another.

40. the method for claim 39, wherein at least a labelled reagent comprises its salt form and/or hydrate forms by following various representative:

Or

Wherein,

^*Expression comprises alternative in due course ¹²C's ¹³C and alternative ¹⁴N's ¹⁵The isotope enrichment position of N;

R ₁Be hydrogen or R ₆

R ₂Be hydrogen or R ₇

Z ' is the leaving group of reaction active groups or reaction active groups;

Wherein,

R ₆And/or R ₇Be alkyl, thiazolinyl, alkynyl, aryl, heteroaryl or aryl alkyl independently of one another.

41. the method for claim 39, wherein at least a labelled reagent comprises its salt form and/or hydrate forms by following various representative:

Or

42. the method for claim 18, wherein said labelled reagent comprises its salt form and/or hydrate forms by formula IV ' representative:

Wherein,

T is 1～10 integer;

R ₁₁Be hydrogen, deuterium, methyl ,-C (H) ₂D ,-C (H) D ₂,-CD ₃, or-R " ';

Z ' is the leaving group of reaction active groups or reaction active groups;

Wherein,

R " ' be H ₂N-R ₉'-, H (R ₁₀) N-R ₉'-, (R ₁₀) ₂N-R ₉'-, HO-R ₉'-, HS-R ₉'-or described compound be can be disconnected to the disconnected connector that couples together with carrier; With

Wherein,

Each R ₉' be alkylidene, alkenylene, alkynylene, arlydene or alkylidene aryl independently of one another; With

Each R ₁₀Be alkyl, thiazolinyl, alkynyl, aryl, heteroaryl or aryl alkyl independently of one another.

43. the method for claim 42, wherein at least a labelled reagent comprises its salt form and/or hydrate forms by following various representative:

Or

Wherein,

^*Expression comprises alternative in due course ¹²C's ¹³C and alternative ¹⁴N's ¹⁵The isotope enrichment position of N; With

Z ' is the leaving group of reaction active groups or reaction active groups.

44. comprise the potpourri of the analyte of at least two kinds of marks, at least two kinds in the analyte of wherein said mark are derived from different samples, the analyte of every kind of mark of wherein said potpourri is by formula I " representative, comprise its salt form and/or hydrate forms:

Wherein,

Y can be substituted or unsubstituted 5,6 or 7 yuan of heterocycles, and wherein said heterocycle comprises by covalent bond and is connected at least one theheterocyclic nitrogen atom on the group J;

J is by formula-CJ ' ₂The group of-representative, wherein each J ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₃,-OR ₃,-SR ₃,-R ₃' OR ₃Or-R ₃' SR ₃

K be by formula-(CK ' ₂) _n-or-((CK ' ₂) _m-X ₃-(CK ' ₂) _m) _pThe group of-representative, wherein n is 2～10 integer, each m is 1～5 integer independently of one another, p is 1～4 integer, each K ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₄,-OR ₄,-SR ₄,-R ₄' OR ₄Or-R ₄' SR ₄

L be by formula-(CL ' ₂) _q-or-((CL ' ₂) _m-X ₃-(CL ' ₂) _m) _pThe group of-representative, wherein q is 1～10 integer, each m is 1～5 integer independently of one another, p is 1～4 integer, each L ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₅,-OR ₅,-SR ₅,-R ₅' OR ₅Or-R ₅' SR ₅

Perhaps

1) R ₁Be hydrogen, deuterium or R ₆, and R ₂Be hydrogen, deuterium or R ₇

Perhaps

2) R ₁And R ₂Be together by formula-(CR ' ₂) _q-or-((CR ' ₂) _m-X ₃-(CR ' ₂) _m) _pThe group of-representative, described group have formed the ring of two nitrogen-atoms of bridge joint, and wherein q is 1～10 integer, and each m is 1～5 integer independently of one another, and p is 1～4 integer, each R ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₆,-OR ₆,-SR ₆,-R ₆' OR ₆Or-R ₆' SR ₆

X ₁Be=O ,=S ,=NH or=NR ₇

Each X ₂Be independently of one another=O or=S;

Each X ₃Be independently of one another-O-or-S-;

Z " be covalently bound analyte;

Wherein,

Each R ₃, R ₄, R ₅, R ₆And/or R ₇Be alkyl, thiazolinyl, alkynyl, aryl, heteroaryl or aryl alkyl independently of one another;

Each R ₃', R ₄', R ₅' and/or R ₆' be alkylidene, alkenylene, alkynylene, arlydene or alkylidene aryl independently of one another; With

Wherein, the described group Y-J that forms report thing part is encoded uniquely at one or more isotope enrichments position, thereby when the key between the remainder of the analyte of the group J of described group Y-J and described mark ruptures in mass spectrometer, generation has the report thing ion of unique qualities, and described report thing ion is identified as each the described sample in labeled reactant source in the analyte of described two or more marks.

45. the potpourri of claim 44, one or more in the wherein said analyte are peptide and/or protein.

46. the potpourri of claim 44, one or more in the wherein said analyte are nucleic acid.

47. the potpourri of claim 44, one or more in the wherein said analyte are lipid and/or steroids.

48. the potpourri of claim 44, one or more in the wherein said analyte are that quality is less than 1500 daltonian micromolecule.

49. the potpourri of claim 44, two or more in the wherein said analyte are different types of analytes.

50. the potpourri of claim 44, wherein the analyte of at least a mark is by formula III " representative, comprise its salt form and/or hydrate forms:

Wherein,

S is 1～5 integer, and t is 1～10 integer;

R ₁Be hydrogen, deuterium or R ₆

R ₂Be hydrogen, deuterium or R ₇

R ₁₁Be hydrogen, deuterium, methyl ,-C (H) ₂D ,-C (H) D ₂,-CD ₃, other alkyl or-R " ';

Z " be covalently bound analyte;

Wherein,

R " ' be H ₂N-R ₉'-, H (R ₁₀) N-R ₉'-, (R ₁₀) ₂N-R ₉'-, HO-R ₉'-or HS-R ₉'-; With

Wherein,

Each R ₆, R ₇And/or R ₁₀Be alkyl, thiazolinyl, alkynyl, aryl, heteroaryl or aryl alkyl independently of one another; With

Each R ₉' be alkylidene, alkenylene, alkynylene, arlydene or alkylidene aryl independently of one another.

51. the potpourri of claim 50, wherein the analyte of at least a mark comprises its salt form and/or hydrate forms by following various representative:

Or

Wherein,

^*Expression comprises alternative in due course ¹²C's ¹³C and alternative ¹⁴N's ¹⁵The isotope enrichment position of N;

R ₁Be hydrogen or R ₆

R ₂Be hydrogen or R ₇

Z " be covalently bound analyte;

Wherein,

R ₆And/or R ₇Be alkyl, thiazolinyl, alkynyl, aryl, heteroaryl or aryl alkyl independently of one another.

52. the potpourri of claim 50, wherein the analyte of at least a mark comprises its salt form and/or hydrate forms by following various representative:

Or

53. the potpourri of claim 44, wherein the analyte of at least a mark is by formula IV " representative, comprise its salt form and/or hydrate forms:

Wherein,

T is 1～10 integer;

R ₁₁Be hydrogen, deuterium, methyl ,-C (H) ₂D ,-C (H) D ₂,-CD ₃, or-R " ';

Z " be covalently bound analyte;

Wherein,

R " ' be H ₂N-R ₉'-, H (R ₁₀) N-R ₉'-, (R ₁₀) ₂N-R ₉'-, HO-R ₉'-or HS-R ₉'-; With

Wherein,

Each R ₉' be alkylidene, alkenylene, alkynylene, arlydene or alkylidene aryl independently of one another; With

Each R ₁₀Be alkyl, thiazolinyl, alkynyl, aryl, heteroaryl or aryl alkyl independently of one another.

54. the potpourri of claim 53, wherein the analyte of at least a mark comprises its salt form and/or hydrate forms by following various representative:

Or

Wherein,

^*Expression comprises alternative in due course ¹²C's ¹³C and alternative ¹⁴N's ¹⁵The isotope enrichment position of N;

Z " be covalently bound analyte.

55. comprise at least a kit according to each compound in the claim 1～17.

56. the kit of claim 55 also comprises other reagent of at least a selection, so that isolabeling is used for the quantitatively mensuration of one or more analytes of two or more different samples by sample being carried out not.

57. the kit of claim 55, wherein said compound are the calibration standard things that comprises the mark of the report thing part with unique qualities.

58. the kit of claim 55, wherein said compound are the labelled reagents that comprises the report thing part with unique qualities.

59. a method comprises:

A) make replacement or unsubstituted diamines and replacement or unsubstituted dicarboxylic acid or anhydride reaction, thereby form the amino acid product that comprises at least one amido link or thioamides key;

B) make described amino acid product and the compound reaction that comprises report thing part, the wherein said compound that comprises report thing part comprise replace or the alkylating acetate part of unsubstituted N-and with the covalently bound nitrogen-atoms of described acetate part; And

C) modify the acid groups or the thio-acid group of described amino acid product, thus form can with the reaction active groups of the functional group reactions of analyte,

Wherein in the product of step (c), comprise at least one isotope enrichment position.

60. the method for claim 59, wherein said method produces the compound by formula I ' representative, comprises its salt form and/or hydrate forms:

Wherein,

Y is 5,6 or 7 yuan of heterocycles, and wherein said heterocycle comprises by covalent bond and is connected at least one theheterocyclic nitrogen atom on the group J;

J is by formula-CJ ' ₂The group of-representative, wherein each J ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₃,-OR ₃,-SR ₃,-R ₃' OR ₃Or-R ₃' SR ₃

K be by formula-(CK ' ₂) _n-or-((CK ' ₂) _m-X ₃-(CK ' ₂) _m) _pThe group of-representative, wherein n is 2～10 integer, each m is 1～5 integer independently of one another, p is 1～4 integer, each K ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₄,-OR ₄,-SR ₄,-R ₄' OR ₄Or-R ₄' SR ₄

L be by formula-(CL ' ₂) _q-or-((CL ' ₂) _m-X ₃-(CL ' ₂) _m) _pThe group of-representative, wherein q is 1～10 integer, each m is 1～5 integer independently of one another, p is 1～4 integer, each L ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₅,-OR ₅,-SR ₅,-R ₅' OR ₅Or-R ₅' SR ₅

Perhaps 1) R ₁Be hydrogen, deuterium or R ₆, and R ₂Be hydrogen, deuterium or R ₇

Perhaps

2) R ₁And R ₂Be together by formula-(CR ' ₂) _q-or-((CR ' ₂) _m-X ₃-(CR ' ₂) _m) _pThe group of-representative, described group have formed the ring of two nitrogen-atoms of bridge joint, and wherein q is 1～10 integer, and each m is 1～5 integer independently of one another, and p is 1～4 integer, each R ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₆,-OR ₆,-SR ₆,-R ₆' OR ₆Or-R ₆' SR ₆

X ₁Be=O ,=S ,=NH or=NR ₇

Each X ₂Be independently of one another=O or=S;

Each X ₃Be independently of one another-O-or-S-;

Z ' is the leaving group of reaction active groups or reaction active groups;

Wherein,

Each R ₃, R ₄, R ₅, R ₆And/or R ₇Be alkyl, thiazolinyl, alkynyl, aryl, heteroaryl or aryl alkyl independently of one another; With

Each R ₃', R ₄', R ₅' and/or R ₆' be alkylidene, alkenylene, alkynylene, arlydene or alkylidene aryl independently of one another.

61. a fragment ions composition, described fragment ions composition is not present in the mass spectrometer after the analyte molecule fracture of isolabeling at least two kinds, and at least a in the analyte molecule of wherein said mark is to be selected from following one of various compound:

With

Wherein said fragment ions is positively charged or electronegative.

62. the fragment ions composition of claim 61, described fragment ions composition has following molecular formula: ¹³C ₆H ₁₃ ¹⁵N ₂ ⁺, ¹³C ₄C ₂H ₁₃ ¹⁵N ₂ ⁺, ¹³C ₃C ₃H ₁₃ ¹⁵N ₂ ⁺, ¹³C ₃C ₃H ₁₃ ¹⁵NN ⁺, ¹³C ₂C ₄H ₁₃ ¹⁵NN ⁺, ¹³CC ₅H ₁₃ ¹⁵NN ⁺, ¹³CC ₅H ₁₃N ₂ ⁺Or C ₆H ₁₃N ₂ ⁺

63. the fragment ions composition of claim 61, described fragment ions composition has following molecular formula: ¹³C ₆H ₁₃ ¹⁵N ₂ ⁺, ¹³C ₄C ₂H ₁₃ ¹⁵N ₂ ⁺Or ¹³C ₃C ₃H ₁₃ ¹⁵N ₂ ⁺

64. potpourri, described potpourri comprises the fragment ions of being derived by the molecule generation fracture of the not isolabeling of analyte, the described fragment ions of the analyte molecule of wherein said not isolabeling produces in mass spectrometer, and the analyte molecule of wherein at least a described not isolabeling is to be selected from following one of various compound:

With

Wherein said fragment ions is positively charged or electronegative.

65. the potpourri of claim 64, wherein at least a fragment ions has following molecular formula: ¹³C ₆H ₁₃ ¹⁵N ₂ ⁺, ¹³C ₄C ₂H ₁₃ ¹⁵N ₂ ⁺, ¹³C ₃C ₃H ₁₃ ¹⁵N ₂ ⁺, ¹³C ₃C ₃H ₁₃ ¹⁵NN ⁺, ¹³C ₂C ₄H ₁₃ ¹⁵NN ⁺, ¹³CC ₅H ₁₃ ¹⁵NN ⁺, ¹³CC ₅H ₁₃N ₂ ⁺Or C ₆H ₁₃N ₂ ⁺

66. the potpourri of claim 64, wherein at least a fragment ions has following molecular formula: ¹³C ₆H ₁₃ ¹⁵N ₂ ⁺, ¹³C ₄C ₂H ₁₃ ¹⁵N ₂ ⁺Or ¹³C ₃C ₃H ₁₃ ¹⁵N ₂ ⁺

67. fragment ions, described fragment ions is fracture takes place in mass spectrometer and to produce by comprising composition with the analyte molecule of amount dystopy mark, and wherein said is to be selected from following one of various compound with at least a in the analyte molecule of measuring the dystopy mark:

With

Wherein said fragment ions is positively charged or electronegative.

68. the composition of the fragment ions of claim 67, it has following molecular formula: ¹³C ₆H ₁₃ ¹⁵N ₂ ⁺, ¹³C ₄C ₂H ₁₃ ¹⁵N ₂ ⁺, ¹³C ₃C ₃H ₁₃ ¹⁵N ₂ ⁺, ¹³C ₃C ₃H ₁₃ ¹⁵NN ⁺, ¹³C ₂C ₄H ₁₃ ¹⁵NN ⁺, ¹³CC ₅H ₁₃ ¹⁵NN ⁺, ¹³CC ₅H ₁₃N ₂ ⁺Or C ₆H ₁₃N ₂ ⁺

69. the composition of the fragment ions of claim 67, it has following molecular formula: ¹³C ₆H ₁₃ ¹⁵N ₂ ⁺, ¹³C ₄C ₂H ₁₃ ¹⁵N ₂ ⁺Or ¹³C ₃C ₃H ₁₃ ¹⁵N ₂ ⁺

70. fragment ions that produces by the following method:

Ionization comprises at least two kinds of not parts of the sample mixture of the analyte molecule of isolabeling in mass spectrometer;

Select the analyte molecule of at least two kinds of described not isolabelings to rupture at selected m/z value place; And

By applying the energy level that dissociates selected analyte molecule is ruptured;

The analyte molecule of wherein at least a described not isolabeling is to be selected from following one of various compound:

With

Wherein said fragment ions is positively charged or electronegative.

71. the composition of the fragment ions of claim 70, it has following molecular formula: ¹³C ₆H ₁₃ ¹⁵N ₂ ⁺, ¹³C ₄C ₂H ₁₃ ¹⁵N ₂ ⁺, ¹³C ₃C ₃H ₁₃ ¹⁵N ₂ ⁺, ¹³C ₃C ₃H ₁₃ ¹⁵NN ⁺, ¹³C ₂C ₄H ₁₃ ¹⁵NN ⁺, ¹³CC ₅H ₁₃ ¹⁵NN ⁺, ¹³CC ₅H ₁₃N ₂ ⁺Or C ₆H ₁₃N ₂ ⁺

72. the composition of the fragment ions of claim 70, it has following molecular formula: ¹³C ₆H ₁₃ ¹⁵N ₂ ⁺, ¹³C ₄C ₂H ₁₃ ¹⁵N ₂ ⁺Or ¹³C ₃C ₃H ₁₃ ¹⁵N ₂ ⁺

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