CN101541774A - Methods, mixtures, kits and compositions pertaining to analyte determination - Google Patents

Abstract

This invention pertains to methods, mixtures, kits and compositions pertaining to analyte determination by mass spectrometry using labeling reagents that comprise a nucleophilic reactive group that reacts with a functional group of an analyte to produce a labeled analyte. The labeling reagents can be used as isobaric sets, mass differential labeling sets or in a combination of isobaric and mass differential labeling sets.

Description

The method, mixture, test kit and the composition that relate to analyte determination

Sub-section titles used herein only for the purpose of tissue, and should not be considered as limiting by any way described theme.

The field

The present invention relates to be undertaken method, mixture, test kit and the composition of analyte determination by mass spectroscopy.

General introduction

The present invention relates to by mass analysis determination and analysis thing.Thereby analyte can be to form any target molecule of stablizing adducts with amido or diazanyl reaction.For example, the reaction active groups of analyte can be carboxylic acid, aldehydes or ketones base, and wherein the adducts that is formed by described aldehydes or ketones base can be reduced and form the stable analyte that is labeled.The non-limiting example of analyte includes but not limited to that biologically important carboxylic acid cpd (for example prostaglandin(PG), lipid acid, carnitine etc.), protein, peptide, carbohydrate, lipid, amino acid, steroid and quality are less than 1500 dalton and comprise other small molecules of suitable reactive functional.Can utilize permission that the unique tag reagent that the analyte in the complex mixture carries out relative and/or absolute quantitation is come the determination and analysis thing.Described labelled reagent can use in groups and be used for the Analysis of Complex sample mixture, wherein labelled reagent can be with (the comprising isomeric with amount allosterism (isobar)) of measuring dystopy (isobaric) and/or comprise the labelled reagent (be different in nature labelled reagent of poor quality, be called " different in nature label of poor quality " in this article again) with unique rough quality.

For example, can utilize the label of isobaric and/or the opposite sex of poor quality to carry out the multiple qualitative and quantitative analysis of biology important compound (for example carboxylic acid cpd such as prostaglandin(PG), lipid acid, carnitine etc.), in some embodiments, (multiple reactionmonitoring MRM) carries out on triple quadrupole bar, linear ion hydrazine instrument can to utilize the multiple reaction monitoring.

With reference to figure 1a, can comprise report thing part, equipoise (or connector) part and reaction active groups part with the labelled reagent in the amount dystopy group, wherein reaction active groups is replaced by described analyte described in the form behind this constituent and analyte response.The labelled reagent of this general formula and the example that is labeled analyte are open in the file for example below: disclosed unsettled jointly and total applicant's U.S. Patent application US 2004-0219685 A1, US 2005-0114042 A1, US2005-0147982 A1, US 2005-0147985 A1, US 2005-0147987 A1, US2005-0148771 A1, US 2005-0148773 A1 and US 2005-0148774 A1.

In some embodiments, can be used for for example analyte in two or more different samples of mark with the labelled reagent of amount dystopy (comprise isomeric with amount dystopy), different labelled reagents in wherein said one group all have identical rough quality but wherein each report thing part can be carried out isotope-codedly uniquely, make that each the report thing part in described group all has unique rough quality.The report thing part that can comprise unique rough quality because all reagent in described group all can have identical rough quality, so equipoise (or connector) part (but be not must) usually also can comprise one or more heavy atom isotopes, quality of " thereby balance " each unique report thing makes that report thing/connector combination of every kind of labelled reagent all has identical rough quality in described group.

In some embodiments, different in nature labelled reagent of poor quality (being different in nature label of poor quality) can be used to for example analyte in two or more different samples of mark, wherein the different labelled reagents in a group all have visibly different quality (promptly with described group in other reagent compare all can have known mass discrepancy).Because all reagent in described group all can have known mass discrepancy, so do not need to make the relative quantity of described labelled reagent fracture with similar analysis thing in quantitative two different samples.Yet in some embodiments, described labelled reagent can rupture.

Discussed in detail as following institute, the compound of various general formulas (comprise and contain the group that has the same structure general formula but also carried out isotope-coded compound) can be prepared into the group with the amount dystopy and/or the opposite sex of poor quality.Usually, group can be used as isobaric marker group or uses as the marker group of the opposite sex of poor quality, but it is also envisioned that as one embodiment of the invention, in same experiment, can unite simultaneously and use with the group of amount dystopy labelled reagent and the group of different in nature labelled reagent of poor quality.

As institute is discussed in detail herein, an example of novel markings reagent (or being labeled analyte) is shown among Fig. 1 b.Though the diagram be do not replace form (except R ₁And R ₂), but be to be understood that described labelled reagent can be replacement or unsubstituted.In the drawings, some keys shows and ruptured, thereby from described labelled reagent or be labeled and discharge described unique report thing part and equipoise part randomly the analyte at least.For with being labeled for the analyte of crossing with amount dystopy labelled reagent mark, at mass spectrograph (usually at MS ⁿIn the analysis, wherein n is for greater than 1 integer) in every kind of unique report thing ion (being sometimes referred to as characteristic ion (signatureion)) of observing all can be used to determine the amount of analyte in sample or the sample mixture.With being labeled for the analyte that different in nature labelled reagent mark of poor quality is crossed, can pass through MS for ¹The relative intensity that is labeled analyte in the analysis is come quantitatively.

Fig. 4 a example the labelled reagent of two groups of 4 kinds of different coding forms, AI to AIV group has the (R for example of the basic structure shown in Fig. 1 b ₁And R ₂Can be hydrogen or methyl independently of one another), wherein asterisk ( ^*) be used to indicate when suitable ¹³The C atom substitutes ¹²The position of C atom, ¹⁵The N atom substitutes ¹⁴The position of N atom or ¹⁸O substitutes ¹⁶The position of O atom.These groups can be used to implement at least 4 heavily experiments (4-plex experiment).Yet, can estimate, by with the further substitution compound of heavy atom isotope, can make different compounds, thereby can carry out more than 4 heavily experiments above 4 kinds.

Usually, labelled reagent, some intermediates of being labeled analyte and described labelled reagent and/or being labeled analyte can be represented with formula I compound (comprising its salt form and/or its hydrate forms):

Wherein Z can be hydrogen or covalently bound analyte, wherein atom or radicals X ₁, R ₁, R ₂, Y, J and K make more detailed description hereinafter.

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

Wherein atom or radicals R ₁, R ₂, Y, J and K make more detailed description hereinafter.In some embodiments, described labelled reagent can use in groups, and wherein these groups comprise isometry and/or isobaric compound, thereby the described analyte that is labeled can be isometry and/or isobaric equally.In some embodiments, described labelled reagent can use in groups, and wherein these groups comprise different in nature labelled reagent of poor quality (labelled reagent that promptly has different rough quality).In some embodiments, use together with amount dystopy (comprising isobaric isomers) labelled reagent and different in nature labelled reagent of poor quality.

Therefore and in some embodiments, being labeled analyte can be with formula I " (comprising its salt form and/or its hydrate forms) represent:

Wherein Z " representative and the covalently bound analyte of labelled reagent, wherein atom or radicals R ₁, R ₂, Y, J and K make more detailed description hereinafter.

As described herein, can prepare two kinds, three kinds, four kinds or more kinds of group, thereby allow 4 heavy or more multiple experiments with amount dystopy and/or different in nature labelled reagent of poor quality.For example, can identify simultaneously and/or quantitatively separately by otherness mark and the analyte in 4 kinds of (or more kinds of) different samples of blended then.For isobaric labelled reagent group, can realize quantitatively by every kind of every kind of relevant unique thing ionic relative abundance of reporting of different labelled reagents in the definite and described dystopy of the amount together group.For quality otherness group, can realize quantitatively by every kind of every kind of relevant relative abundance that is labeled analyte of different labelled reagents in the definite and described opposite sex group of poor quality.

Therefore, embodiment of the present invention are particularly suitable for the multiple analysis (multiplex analysis) of complex sample mixture.For example, embodiments more of the present invention can be used in Proteomic analysis and/or genome analysis and the cognation research relevant with Proteomic analysis and/or genome analysis.Embodiments more of the present invention also can be used for the small molecules analysis, such as being used for carnitine, carbohydrate, lipid, steroid, VITAMIN, prostaglandin(PG), lipid acid and/or amino acid analysis.Can use the mensuration (referring to for example laid-open U.S. Patents application US 2005-0208550A1) and the multiple control experiment that include but not limited to time course research, biomarker analysis, multiplexed protein matter group analysis, the experiment of multidimensional protein identification techniques (mudpit experiment), affinity capture (affinity pull-down), posttranslational modification (PTM) with the experimental analysis of amount dystopy and/or different in nature reagent of poor quality.

Description of drawings

It will be understood to those of skill in the art that the following stated accompanying drawing is only presented for purposes of illustration.These accompanying drawings are not the scope that is intended to limit by any way the present invention's instruction.

Fig. 1 a is illustrating of labelled reagent or the key element that is labeled analyte and some fracture characteristics.

Fig. 1 b be exemplary based on N methyl piperazine labelled reagent or be labeled the general key element of analyte and illustrating of some fracture characteristics.

Fig. 1 c be the general formula that identifies be labeled the general key element of analyte and illustrating of some fracture characteristics.

Fig. 2 is the illustrating of general formula (being structure A and structure B) of two kinds of different labelled reagents.

Fig. 3 utilizes according to the possible breaking property of the analyte (be expressed as and be labeled analyte A " and B ") of the labelled reagent institute mark of structure A and B and illustrating by the possible segmental quality of its generation.

Fig. 4 a is the illustrating of group of two kinds of possible same amount dystopy labelled reagents (respectively based on structure A and structure B), and wherein every kind of labelled reagent produces same report thing ionic group.

Fig. 4 b is illustrating of possible the structure of the thing ion of report described in Fig. 4 a 114-117 and possible structure that noncoding (i.e. " cold ") reports thing ion 113.

Fig. 5 a and 5b are the illustrating of group of two kinds of possible of poor quality different in nature labelled reagents (respectively based on structure A and structure B).

Fig. 6 a and 6b utilize the exemplary compounds of structure A and structure B to come the illustrating of method of labelled analyte.

All clear and definite all being incorporated herein by reference of the whole documents quoted among the application and analogous material (including but not limited to patent, patent application, article, books, collected works and internet page) and be used for any and whole purposes, no matter and the form of these documents and analogous material how.

Definition

For the purpose of explaining this specification, will use following definitions; As long as when suitable, the term of using with odd number also will comprise its plural form, and vice versa. For the purpose of explaining this specification and related right claim thereof, in any following definitions and any other file (be included as autotelic any by with reference to the file of incorporating this paper into) in the inconsistent situation of this word usage, will be always with the following standard that is defined as, unless explicitly point out opposite implication (for example when explaining the file of initial this term of use). Herein the usage of "or" mean " and/or ", unless otherwise indicated or use " and/or " in the situation about obviously not conforming to. Limit without quantity herein or mean when using " one " (" a ") " one or more ", unless otherwise indicated or in " one or more " obvious inappropriate situation of use. " comprise ", " comprising ", " containing " be used interchangeably, be not intended to limit. In addition, in the description of one or more embodiments, used in the situation that term " comprises ", it will be understood to those of skill in the art that under some concrete conditions, as an alternative, described embodiment can use language " basically by ... form " and/or " by ... form " describe. It is also understood that in some embodiments the order of step or to carry out the order of some action unimportant is as long as instruction of the present invention still can be implemented. And, in some embodiments, can carry out simultaneously two or more steps or action.

A.) " analyte " used herein refers to measurable target molecule. The non-limiting example of analyte includes but not limited to that protein, peptide, (DNA or RNA), carbohydrate, lipid, amino acid, steroids, vitamin, prostaglandin, aliphatic acid, carnitine and molecular weight are less than other little molecule of 1500 dalton (Da). Analyte or to contain the source of sample of analyte unrestricted is because they can be from any source. Analyte can be natural or synthetic. Analyte or the non-limiting example in source that contains the sample of analyte comprise cell or tissue or its culture (or subculture). The non-limiting example in analyte source includes but not limited to rough cell lysate or treated cell lysate, body fluid, tissue extract or from the fraction (or part) of separation process, described separation process separates or capillary electrophoresis separation such as chromatographic isolation, one dimension electrophoretic separation, two dimensional electrophoresis. Body fluid includes but not limited to blood, urine, ight soil, spinal fluid, brain liquid, amniotic fluid, lymph liquid or from the fluid of glandular secretion thing. For treated cell lysate, we mean also to process cell lysate except the required processing of cell lysis, thereby collected material is carried out more multiprocessing. For example, described sample can be the cell lysate that contains one or more analytes, thereby described analyte is by process the peptide that cell lysate digestion precursor peptide and/or protein form with one or more proteolytic enzymes.

B.) except when when using context and clearly illustrate that not to be that in addition, " ester " all refers to ester and/or thioesters to this meaning (for example when the other described concrete structure of reference).

C.) " fracture " used herein refer to the fracture of covalent bond.

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

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

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

G.) " isotope enrichment " used for compound herein refer to enrichment one or more heavy atom isotopes (for example stable isotope such as deuterium (' D '),¹³C、 ¹⁵N、 ¹⁸O、 ³⁷Cl or⁸¹Br) compound (for example labelled reagent). In some embodiments, (for example can also use unsettled isotope¹⁴C or³H). The amount that " enrichment " means heavy atom isotope has surpassed natural isotopic abundance. In a plurality of embodiments, the compound of described isotope enrichment can comprise 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 site.

Because isotope enrichment is not 100% effective, so can have impurity in the sample of compound, it is for the state of less enrichment and have lower quality. Equally, owing to excessive enrichment (enrichment of for example not expecting) and owing to natural isotopic abundance, in the sample of compound, can there be the impurity with larger quality. In some embodiments, the isotopic purity that the heavy atom isotope of every species diversity mark can at least 80% is present on the isotope enrichment site. In some embodiments, the isotopic purity that the heavy atom isotope of every species diversity mark can at least 93% is present on the isotope enrichment site. In some embodiments, the isotopic purity that the heavy atom isotope of every species diversity mark can at least 96% is present on the isotope enrichment site. In some embodiments, the purity that the heavy atom isotope of every species diversity mark can at least 98% is present on the isotope enrichment site.

H.) " isotope enrichment site " used herein refers to that heavy atom isotope (for example substitutes light form atom in the compound¹³C substitutes¹²C、 ¹⁸O substitutes¹⁶O、 ¹⁵N substitutes¹⁴N or deuterium instead of hydrogen) the position.

When i.) using for compound herein, " gently " refers to the not compound of enrichment heavy atom isotope. When using for atom herein, " gently " refers to the minimum quality isotope of this atom. When using for compound herein, " weight " compound of at least a heavy atom isotope that referred to enrichment. When using for atom herein, " weight " refers to the heavy Indium isotopes of this atom.

J.) " labelled reagent " used herein refers to be suitable for the structure division of mark analyte to be determined. Terms tag thing (label) is with term tag (tag) and indicate that (mark) and other equivalent terms and phrase are synonyms. For example, being labeled analyte can also be called by the label analyte or by the sign analyte. Therefore the derivatives of term " label ", " label ", " quality tab ", " mark " and these terms all is of equal value and interchangeable, refers to suitable mark or the structure division of mark analyte to be determined. Sometimes labelled reagent can be described as tagging reagents, mass tagging reagents or referred to as quality tab (for example different in nature label of poor quality).

K.) " natural isotopic abundance " used herein refers to the level (or distribution) of one or more heavy isotopes of finding at natural inherent advantage degree (prevalence) based on isotope in compound. For example, by the native compound that obtains in the living plant material usually can comprise with respect to¹²C's about 1.08%¹³C。

I.) used herein is to have the rough quality of identical name and structurally and chemically undistinguishable compound (except the isotopic content of heavy atom isotope and/or distributing) with amount dystopy thing (isobar). " chemically undistinguishable " means describedly to comprise same general chemical constitution (if not considering the distribution of heavy atom isotope) and have substantially the same chemical reactivity and separating property with amount dystopy thing.

M.) " holder " used herein, " solid support ", " solid phase carrier " or " resin " mean any solid phase material. Solid support comprises that term is such as " holder ", " synthetic holder ", " solid phase ", " surface ", " film " and/or " holder ". Solid support can be made of organic polymer, such as polystyrene, polyethylene, polypropylene, polyvinyl fluoride, polyoxyethylene (polyethyleneoxy) and polyacrylamide with and copolymer and graft. Solid support can also be inorganic, such as glass, silica, controlled pore glass (CPG) or anti-phase silica. The structure of solid support can be the form on pearl, ball, particulate, particle, gel, film or surface. The surface can be the plane plane, basic or nonplanar. Solid support can be porous or non-porous, and can have expansion characteristics or unexpansive characteristic. Solid support can make up with the form of hole (well), hole (depression) or other container, vessel, architectural feature (feature) or position (location). A plurality of solid supports can be set to the array on the diverse location, and it is addressable to automatically sending of reagent, perhaps pass through detection method and/or equipment and addressing.

N.) " sample or its fraction " used herein or " sample fraction " can be used for referring to the fraction of sample. The fraction of sample can be only fraction by sample thief produce, perhaps by causing sample classification to produce for the separation process of two or more fractions. Unless point out in addition in the content of specification, otherwise these phrases are of equal value and interchangeable, refer to the sample fraction (or part) of any internus.

O.) " characteristic ion " used herein (signature ion) and " report thing ion " are interchangeable, all refer to by report thing part by labelled reagent or be labeled the report thing ion of the unique qualities that the fracture of analyte produces. Characteristic ion or report thing ion can be used to differentiate the unique tag reagent for labelled analyte, and described characteristic ion or report thing ion are analyzed (or MS at MS/MSⁿAnalyze) in peak intensity can be associated with the amount that is labeled analyte that exists in the institute analytic sample. Characteristic ion used herein or report thing ion only are called the report thing sometimes. Report thing part used herein also only is called the report thing sometimes. Should be appreciated that the report thing partly refers to labelled reagent, is labeled the group that analyte or its fragment link to each other, report thing ion refer to when the described report thing of connection partly with labelled reagent, the fragment ions that produces when being labeled the bond fission of analyte or its fragment. Therefore, use the context of " report thing " word can indicate its purpose implication. It is also understood that phrase " unique report thing part " and " the report thing part with unique qualities " are of equal value and interchangeable, " unique report thing ion " is of equal value and interchangeable with " the report thing ion with unique qualities ".

P.) term used herein " salt form " comprises the mixture of the salt of the salt of compound or compound. In addition, the amphoteric form of compound also is included in the term " salt form ". Salt with compound of amine or other base groups can be by such as obtaining with suitable organic acid or inorganic acid (such as hydrochloric acid, hydrobromic acid, acetic acid, perchloric acid etc.) reaction. The compound that contains quaternary ammonium group also can contain pair anion, such as chlorion, bromide ion, iodide ion, acetate, perchlorate etc. Salt with compound of carboxylic acid or other acidic functionality can make by described compound and suitable alkali (for example hydroxide bases) are reacted. Therefore, the salt of acidic functionality can have counter cation, such as sodium, potassium, magnesium, calcium etc.

Q.) " synthetic compound " used herein refers to comprise the compound that the natural approach of operation produces by operating process. Therefore, can utilize synthesising chemical technology to produce synthetic compound. Yet, " synthetic compound " used herein also is intended to comprise the compound that for example produces by enzyme method, described enzyme method for example comprises feeds the compound of raising isotope enrichment 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 can change the compound of these isotope enrichments.

R.) " synthetic enrichment " used herein refer to that thus the synthetic or natural process of operation produces synthetic compound, such as the labelled reagent of described isotopic enrichment herein or the intermediate of described labelled reagent.

S.) be recognized that the quality of atom or molecule can be got approximate, one of normal approximate 1/10th or percentage that arrives immediate integer atomic mass unit or immediate atomic mass unit." rough quality " used herein refers within the specific limits absolute mass and Approximation Quality, the isotropic substance of the approaching different atomic types of functional quality in described scope, whether make that they are of equal value for the purpose of the quality of described report thing of balance and/or connector part (making that the group at isometry and/or isobaric labelled reagent is identical with the rough quality of reporting thing/connector combination described in the test kit) on function, no matter and used different isotropic substance types can be detected in qualitative minimum difference.

For example, the common isotopic rough quality of oxygen is 16.0 (actual mass 15.9949) and 18.0 (actual mass 17.9992), the common isotopic rough quality of carbon is 12.0 (actual mass 12.00000) and 13.0 (actual mass 13.00336), and the common isotopic rough quality of nitrogen is 14.0 (actual mass 14.0031) and 15.0 (actual mass 15.0001).Although these values are approximation, it will be appreciated by those skilled in the art that if use at the isotopic enrichment position in the marker in a group ¹⁸The O isotropic substance then contains in this group ¹⁶In the different markers of O, 2 mass units that have more (surpassing rough quality and be the value of 16.0 oxygen isotope) can be introduced two carbon by for example other in described marker is local ¹³The C atom substitutes two ¹²C atom, two ¹⁵The N atom substitutes two ¹⁴N atom or even one ¹³C atom and one ¹⁵The N atom substitutes ¹²C and ¹⁴N is with compensation ¹⁸O and being compensated.Like this, described can have identical rough quality with two different markers in the amount dystopy group, because in all markers of described group or test kit, use 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) realize that quality increases by 2 dalton, actual mass difference minimum between them can't counteract this analysis self.

Obviously, in the structure distribution of identical heavy atom isotope be not when the group that produces with amount dystopy and/or different in nature labelled reagent of poor quality the factor of unique consideration.The same amount dystopy or the different in nature marker of poor quality that the heavy atom isotope type hybrid can be had desired rough quality with generation.Like this, to the selection (combination) of heavy atom isotope with and distribute all and can when preparation can be used for the same amount dystopy of embodiment of the present invention and/or different in nature labelled reagent of poor quality, consider.

T.) term used herein " alkyl " refers to it can is complete saturated straight or branched C ₂-C ₈Hydrocarbon or ring-type C ₃-C ₈Hydrocarbon (being that cycloalkyl is such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cyclohexylmethylene).Term used herein " alkyl " refers to be substituted or unsubstituted group.In some embodiments, term " alkyl " also be intended to refer on the alkyl chain wherein one or more methylene radical can by heteroatoms (as-O-,-Si-or-S-) displaced those compounds.In some embodiments, alkyl can be can be by complete saturated straight or branched C ₂-C ₆Hydrocarbon or ring-type C ₃-C ₆Hydrocarbon.

U.) term used herein " alkylidene group " refers to the alkyl chain or the cyclic alkyl of straight or branched, and it comprises at least two tie points being connected with at least two structure divisions ({ CH for example ₂}-(methylene radical) ,-{ CH ₂CH ₂}-(ethylidene) },

Deng, tie point represented in its bracket).When using in this article, term " alkylidene group " refers to be substituted or unsubstituted group.In some embodiments, term " alkyl " also be intended to refer on the alkyl chain of alkylidene group wherein one or more methylene radical (if there is) can by heteroatoms (as-O-,-Si-or-S-) those compounds of institute's alternate.In some embodiments, alkylidene group can be C ₁-C ₁₀Hydrocarbon.In some embodiments, alkylidene group can be C ₂-C ₆Hydrocarbon.

V.) term used herein " thiazolinyl " refers to comprise the straight chain or the ramose C of one or more pairs of keys ₂-C ₈Hydrocarbon polymer or ring-type C ₃-C ₈Hydrocarbon polymer.When using in this article, term " thiazolinyl " refers to be substituted or unsubstituted group.In some embodiments, term " thiazolinyl " also be intended to represent in the alkyl chain of thiazolinyl one or more methylene radical (if any) by heteroatoms (as-O-,-Si-or-S-) alternate compound.In some embodiments, thiazolinyl can be straight chain or the ramose C that comprises one or more pairs of keys ₂-C ₆Hydrocarbon polymer or ring-type C ₃-C ₆Hydrocarbon polymer.

W.) term used herein " alkenylene " refers to comprise the thiazolinyl of two tie points of at least two structure divisions of connection.When using in this article, term " alkenylene " refers to be substituted or unsubstituted group.In some embodiments, term " alkenylene " also be intended to represent in the alkyl chain of alkenylene one or more methylene radical (if any) by heteroatoms (as-O-,-Si-or-S-) alternate compound.

X.) term used herein " alkynyl " refers to comprise one or more triple-linked straight chains or ramose C ₂-C ₈Hydrocarbon polymer or ring-type C ₃-C ₈Hydrocarbon polymer.When using in this article, term " alkynyl " refers to be substituted or unsubstituted group.In some embodiments, term " alkynyl " also be intended to represent in the alkyl chain of alkynyl one or more methylene radical (if any) by heteroatoms (as-O-,-Si-or-S-) alternate compound.In some embodiments, alkynyl can be to comprise one or more triple-linked straight chains or ramose C ₂-C ₆Hydrocarbon polymer or ring-type C ₃-C ₆Hydrocarbon polymer.

Y.) term used herein " alkynylene " refers to comprise the alkynyl of two tie points of at least two parts of connection.When using in this article, term " alkynylene " refers to be substituted or unsubstituted group.In some embodiments, term " alkynylene " also be intended to represent in the alkyl chain of alkynylene one or more methylene radical (if any) by heteroatoms (as-O-,-Si-or-S-) alternate compound.

Z.) term used herein " aliphatics " refers to any straight chain, branch or the cyclic alkyl, thiazolinyl and the alkynyl part that define as mentioned.When using in this article, term " aliphatics " refers to it can is to replace or unsubstituted group.

Aa.) term used herein " aryl " (independent or as the part (for example arylalkyl etc.) of other structure divisions) refers to carbocyclic aromatic group, for example phenyl.Aryl also comprises many cyclophanes of condensed ring ring system, wherein carbocyclic ring aromatic ring and another carbocyclic ring aromatic ring condense (for example 1-naphthyl, 2-naphthyl, 1-anthryl, 2-anthryl etc.), and perhaps carbocyclic ring aromatic ring and one or more carbocyclic ring non-aromatic ring condense (for example tetrahydrochysene naphthylidene, indane etc.).Term used herein " aryl " refers to it can is to replace or unsubstituted group.

Ab.) term used herein " heteroaryl " refers to comprise 1,2,3 or 4 heteroatomic aromatic heterocycle, and described heteroatoms is selected from nitrogen, sulphur and oxygen independently of one another.Term used herein " heteroaryl " refers to it can is to replace or unsubstituted group.Heteroaryl can encircle (as cycloalkyl, aryl or heteroaryl ring) with one or two and condense.The tie point of heteroaryl and molecule can be on heteroaryl, cycloalkyl, Heterocyclylalkyl or aryl rings, and described heteroaryl can connect by carbon or heteroatoms.The example of heteroaryl comprises imidazolyl, pyrryl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, quinolyl, isoquinolyl, indazolyl, indolizinyl (indolizinyl), imidazopyridyl, pyrazolyl, triazolyl, tetrazyl, indyl, tetrahydro indole base, azaindolyl (azaindolyl), imidazopyridyl, quinazolyl, purine radicals, pyrrolo-[2,3] pyrimidyl or pyrazolo [3,4] pyrimidyl, each in them all can randomly be substituted.

Ac.) term used herein " arylidene " refers to comprise the aryl or the heteroaryl (for example phenylene etc.) of at least two tie points that link to each other with at least two structure divisions.Arylidene and carbocyclic ring non-aromatic ring condensed tie point can be on aromatic ring or non-aromatic rings.Term used herein " arylidene " refers to it can is to replace or unsubstituted group.

Ad.) term used herein " arylalkyl " refers to the aryl or the heteroaryl that are connected with another structure division by the alkylidene group connector.Term used herein " arylalkyl " refers to it can is to replace or unsubstituted group.In some embodiments, term " arylalkyl " also be intended to represent in the alkyl chain of arylalkyl one or more methylene radical (if any) by heteroatoms (as-O-,-Si-or-S-) alternate compound.

Ae.) term used herein " aryl alkylene " refers to have the arylalkyl of at least two tie points that link to each other with at least two structure divisions.Second tie point can be aromatic ring or alkylidene group.Term used herein " aryl alkylene " refers to it can is to replace or unsubstituted group.In some embodiments, term " aryl alkylene " also be intended to represent in the alkyl chain of aryl alkylene one or more methylene radical (if any) by heteroatoms (as-O-,-Si-or-S-) alternate compound.When aryl alkylene take place to replace, substituting group can be on the aromatic ring of this aryl alkylene or any one or two in the alkylidene group structure division.

Af.) term used herein " optional replacement " and " replacement or unsubstituted " are that be equal to and interchangeable.The suitable substituent of any alkyl, alkylidene group, thiazolinyl, alkenylene, alkynyl, alkynylene, aryl, arylalkyl, arylidene, heteroaryl or aryl alkylene includes any substituting group stable under the employed reaction conditions of embodiment of the present invention.The limiting examples of suitable substituent can comprise that alkyl (as methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, isobutyl-, sec-butyl, the tertiary butyl, cyclohexyl etc.), haloalkyl are (as trifluoromethyl, 2,2,2-trifluoroethyl etc.), alkoxyl group (as methoxyl group, oxyethyl group etc.), aryl (as phenyl), arylalkyl (as benzyl), nitro (NO ₂), cyano group (CN), quaternary nitrogen atoms or halogen radical (as fluorine, chlorine, bromine and/or iodine).

In addition, the part of alkyl, alkylidene group, thiazolinyl, alkenylene, alkynyl, alkynylene, aryl, arylalkyl, arylidene, heteroaryl or aryl alkylene can be randomly by=O or=S replaces.

Ag.) term used herein " active ester " refer under alkaline condition, be easy to amine, pure and mild some thiol reactant so that the compound of acid amides, ester and thioesters to be provided respectively.Also can be with reference to Leo APaquette, Encyclopedia of Reagents for Organic Synthesis, the 2nd volume, JohnWiley and Sons, New York, 1995, it is the evidence of the clear and definite term in the organic chemistry filed as active ester.

Ah.) term used herein " heterocycle " refers to comprise any ring shaped molecule structure of at least two kinds of different elements in ring.Also can be with reference to Oxford Dictionary of Biochemistry andMolecular Biology, Oxford University Press, Oxford, 1997, it is the evidence of the clear and definite term in the organic chemistry filed as heterocycle.

Ai.) term used herein " leavings group " refers in substitution reaction or replacement(metathesis)reaction any charged or uncharged atom or the group of leaving away from the residue that is considered to reaction substrate or major portion.Also can be with reference to Oxford Dictionary of Biochemistry and MolecularBiology, Oxford University Press, Oxford, 1997, it is the evidence of the clear and definite term in the organic chemistry filed as leavings group.

Aj.) term used herein " blocking group " thus refer to molecule in functional group reactions and combine with it and to prevent that this functional group from participating in the chemical group of the subsequent reactions of this molecule, described group can be removed subsequently, thereby bears unprotected functional group again.Also can be with reference to Oxford Dictionary ofBiochemistry and Molecular Biology, Oxford University Press, Oxford, 1997, it is the evidence of the clear and definite term in the organic chemistry filed as blocking group.

Specific embodiments

Should be appreciated that hereinafter the discussion in " general introduction " joint can relate to some or all in the multiple embodiments of the present invention.

I. general introduction

Labelled reagent:

Labelled reagent can comprise report thing part, equipoise part (or connector part) and reaction active groups, and (Fig. 1 a).Labelled reagent can with analyte response, be labeled analyte thereby produce.In some embodiments, described labelled reagent is organized in groups.In some embodiments, described group can be isometry and/or isobaric.In some embodiments, described group can be the opposite sex of poor quality.In some embodiments, described group can be the opposite sex isobaric and/or isomeric and of poor quality.

In some embodiments, described labelled reagent can have as shown in Figure 2 general structure A or B.The labelled reagent of general structure A and B can form structure A as shown in Figure 3 with analyte response " and B ".In some embodiments, the described analyte that is labeled can rupture in mass spectrograph.Fragment and respective quality thereof that some are possible are found among Fig. 3.Two possible groups of the isobaric labelled reagent of general structure A and B are found among Fig. 4 a, and possible report thing ion sees among Fig. 4 b.Two groups of different in nature labels of poor quality based on general structure A and B are found among Fig. 5 a and Fig. 5 b.Fig. 6 a and Fig. 6 b are for example clear to utilize the labelled reagent of general structure A and B that analyte is carried out mark.

Reaction active groups:

The reaction active groups of the labelled reagent that in the embodiment of method of the present invention, mixture, test kit and/or composition, uses (sometimes use abbreviation " RG " expression) can be can with the nucleophilic group of one or more functional group reactionses of one or more reactive behavior analytes in the sample.Should be appreciated that in some embodiments, can think that described reaction active groups comprises atom or the group relevant with connector (equipoise).

Be to be understood that, when described reaction active groups during by following some concrete structure part representatives, analyte can link to each other with connector (equipoise) by one or more extra atoms or group, and described atom or group can be thought or not think the part of described connector (equipoise).

Described 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 does not have the reactive behavior analyte, and perhaps it can carry out under the situation that has the reactive behavior analyte.In some embodiments, described reaction active groups can be removed blocking group by original position in position and forms.Therefore, any reagent that exists or newly produce of the derivatize of analyte of can realizing by the reaction of nucleophilic thing and/or close electric thing all is encompassed in the embodiment of method of the present invention, mixture, test kit and/or composition.

Reaction active groups at labelled reagent is under the situation of nucleophilic thing, and it can react with the suitable electrophilic group of analyte.A plurality of suitable nucleophilic groups and electrophilic group are to being known and being usually used in chemistry and the biochemical field.Comprise and suitable can be described in Pierce Life Science ﹠amp with the non-limiting example of the reagent of analyte (biological example is learned and gone up important carboxylic acid cpd (for example prostaglandin(PG), lipid acid, carnitine etc.), protein, peptide, carbohydrate, lipid, steroid or quality less than 1500 daltonian other small molecules) link coupled nucleophilic group; AnalyticalResearch Products Catalog ﹠amp; Handbook (a Perstorp Biotec Company), Rockford, IL 61105, among the USA.Other suitable reagent is well-known in the art, and can obtain from how tame supplier (such as Sigma-Aldrich) be commercial.

In some embodiments, the reaction active groups of labelled reagent can be the nucleophilic thing, such as amino or diazanyl.In some embodiments, described nucleophillic attack active group can be aminoalkyl groups or alkyl hydrazine group.

Report thing part:

The report thing of institute's applying marking reagent part in the embodiment of the present invention (using abbreviation " RP " expression sometimes) can be the group with measurable unique qualities (perhaps mass-to-charge ratio in the mass spectrograph).Therefore, in some embodiments, isometry and/or every kind of report thing part measuring together in the dystopy labelled reagent group all have unique rough quality, and the individual features ion of every kind of labelled reagent differs from one another in organizing.

Different report thing parts can comprise one or more heavy atom isotopes, to realize the rough quality of its uniqueness.For example, there is following isotropic substance: carbon ( ¹²C, ¹³C and ¹⁴C), nitrogen ( ¹⁴N and ¹⁵N), oxygen ( ¹⁶O and ¹⁸O), sulphur ( ³²S and ³⁴S) or hydrogen (hydrogen, deuterium and tritium), and they can be used for preparing the different groups of report thing part.They are not restrictive, because also can use other light atom and heavy atom in report thing part.The basic parent material that is suitable for preparing report thing part that comprises light atom and heavy atom isotope can derive from a plurality of commercial source, Cambridge Isotope Laboratories for example, and Andover, MA (consults Www.isotope.com" basic parent material " tabulation) and Isotec (branch of Sigma-Aldrich).Cambridge Isotope Laboratories and Isotec also can prepare needed compound according to the synthetic contract of customization, and the source is the same.

Report thing part can comprise the fixed electric charge, perhaps can ionization in analytic process.Because report thing part can comprise fixed charge or can be ionized, therefore described labelled reagent can salt (or mixture of salt) or zwitterionic isolated in form or is used for labeled reactant activation analysis thing.The ionization of report thing part (or report thing ion) helps in mass spectrograph it being measured.Therefore, being labeled the report thing that exists in the analyte partly can be used as fragment ions (being sometimes referred to as characteristic ion (or report thing ion)) and measures.

After the ionization, characteristic ion (promptly reporting the thing ion) can comprise one or more clean positive charges or negative charge.Therefore, report thing ion can comprise one or more acidic-groups and/or basic group, because these groups can be easy to ionization in mass spectrograph.For example, report thing part can comprise one or more basic nitrogen atoms (positively charged) and/or one or more ionized acidic-group, for example carboxylic acid group, sulfonic group or phosphate (electronegative) are as long as exist when balance more than the basic group of or other number or acidic-group so that this report thing partly produces the report thing ion with clean positive charge or negative charge.The non-limiting example that comprises the report thing part of at least one basic nitrogen comprises and replacing or the unsubstituted compound that contains morpholine, piperidines or piperazine.

Unique report thing part can be associated with the purpose sample, thereby utilizes one or more analytes in this sample of report thing part mark of described uniqueness.Like this, can will report thing information (generally in mass spectrograph, detecting) partly about uniqueness and be associated about information a kind of in the described sample or whole analytes by characteristic ion (promptly reporting the thing ion).

Yet, when measuring characteristic ion, unique report thing part might not with the analyte physical connection.On the contrary, the ion that unique rough quality of characteristic ion can be for example be labeled analyte in fracture is measured in second mass analysis in the series connection analyser with after producing sub-fragment ions and characteristic ion.

The characteristic ion of measuring can be used for the sample of identifying that institute's determination and analysis thing is derived from.In addition, with respect to the amount of other characteristic ions or with respect to the amount of the characteristic ion relevant with the calibration standard thing (calibration standard thing for example be present in the described sample for expection and with the analyte of specificity report thing part mark), the amount of the characteristic ion of described uniqueness (being often expressed as concentration and/or amount) can be used for measuring the relative quantity and/or the absolute magnitude (being often expressed as concentration and/or amount) of analyte in described sample or the described a plurality of sample (for example being used to form the sample of sample mixture).In some embodiments, be not to use the internal calibration standard substance, but can be based on various features ionic peak intensity and calibration curve are compared and carry out absolute quantitation.Therefore, can be with information (for example amount of one or more analytes in the specific sample) and the report thing part correlation connection that is used for every kind of specific sample of mark.When also determining the identity of described analyte, this information can be associated with relating to different characteristics ionic information, thus every kind of identity and amount that is labeled analyte in auxiliary definite one or more samples.

In some embodiments, report thing part can comprise with replacement or unsubstituted N-alkylation acetate part in the covalently bound nitrogen-atoms of mesomethylene carbon, wherein said replacement or unsubstituted the mesomethylene carbon carbonyl of carboxylic acid (or thiocarboxylic acid) group (rather than in this acetate part) are the parts of this report thing.Therefore, in some embodiments, carboxylic acid (or thiocarboxylic acid) base can be used for connecting report thing and connector, but does not think to report the part of thing.Described nitrogen-atoms can be by one, two or three group alkylations.For example, the part that comprises nitrogen-atoms can be the primary amine that replaces, for example methyl, ethyl or propyl group, or the secondary amine that replaces, for example dimethylamine, diethylamine, di-n-propyl amine or diisopropylamine.Therefore, for example, report thing part " RP " can be by following formula X ¹, X ², X ³, X ⁴, X ⁵, X ⁶, X ⁷, X ⁸Or X ⁹Expression, report that wherein thing part " RP " is the boundary with the bracket:

Report thing part can be 5,6 or 7 element heterocycles, it comprises the covalently bound theheterocyclic nitrogen atom of mesomethylene carbon with replacement or unsubstituted N-alkylation acetate part, described replacement or unsubstituted N-alkylation acetate partly link to each other with analyte (directly or indirectly) by its carbonyl carbon, and wherein said replacement or unsubstituted mesomethylene carbon (rather than carbonyl carbon of carboxylic acid group) are the parts of this report thing.Described heterocycle can be fragrance or non-fragrance.Therefore, described report thing part can be by formula Y-J representative, and wherein Y group can be represented 5,6 or 7 element heterocycles, and the J group can be represented in described replacement or the unsubstituted acetate part and replace or unsubstituted methylene radical.Described heterocycle can be replacement or unsubstituted.For example, the substituting group of this heterocyclic moiety can comprise alkyl, alkoxyl group and/or aryl.Substituting group can comprise and is suitable for protection or unprotected group that analyte is connected with upholder, for example amine, hydroxyl or thiol group (see figure 6).Described heterocycle also can comprise extra heteroatoms, for example one or more silicon, nitrogen, oxygen and/or sulphur atom.Therefore, for example, report thing part " RP " can be by following formula X ¹⁰, X ¹¹, X ¹², X ¹³, X ¹⁴, X ¹⁵, X ¹⁶, X ¹⁷, X ¹⁸, X ¹⁹, X ²⁰, X ²¹, X ²², X ²³, X ²⁴, X ²⁵Or X ²⁶Represent, report that wherein thing part " RP " is the boundary with the bracket:

Can partly select the report thing, so that inferior fracture (sub-fragment) does not take place under the usual conditions that analyte is analyzed for it.In order to eliminate any doubt, this be optional feature of (and nonessential).For example, can select, significant inferior fracture does not take place under the dissociation energy condition that is labeled the analyte fracture in mass spectrograph so that it makes to the report thing." significant inferior fracture not taking place " and mean when successfully mensuration is labeled analyte, is difficult to maybe can not detect the report thing fragment that is higher than ground unrest.

In some embodiments, can be intentionally the rough quality of report thing ionic is chosen to and is different from the analyte seeking to measure or the segmental quality of any expectation (i.e. " dead zone ") of this analyte.For example, when analyte is protein or peptide, can select to report that the rough quality of thing ionic makes it be different from any natural amino acid or peptide or its and estimates fragment.This can help analyte determination, because for analyte, does not have any possibility component with identical quality in the sample, and this can improve the degree of confidence of any analytical results.For peptide, can estimate that the example of the mass range that background is extremely low is found in table 1.

Table 1: the possibility " dead zone " of selecting the marker fragment ions m/z relevant with peptide analysis

M/z starting point-terminal point
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

Described report thing part can be non-polymeric.Can select to report that the thing part shows the characteristic ion of its quality less than the m/z of 250 atomic mass units (amu) with generation.Can select to report that the thing part shows the characteristic ion of its quality less than the m/z of 200 atomic mass units (amu) with generation.Can select to report that the thing part shows the characteristic ion of its quality less than the m/z of 150 atomic mass units (amu) with generation.These small molecules can easily be measured in second mass analysis, do not contain other component that has same consistent quality (coincident mass) in the sample with described first mass analysis in described second mass analysis.In this case, can carry out second mass analysis, generally in tandom mass spectrometer, carry out (perhaps, for example in the single-stage instrument, passing through post-source decay) and carry out the selected ion of measuring in first mass analysis.Be used for possible fracture and further mass analysis owing to can select the ion with specific mass-to-charge ratio from first mass analysis, therefore unselected ion does not carry out second mass analysis in first mass analysis, thereby does not pollute the spectrum of second mass analysis.And the linearity of mass spectrometric sensitivity and detector (being used for quantitatively) can be quite sane in this inferior quality scope.In addition, the current state of mass spectrometry art can allow to obtain less than 1 daltonian baseline mass resolution in this mass range.Owing to all these reasons, the report thing processing with above-mentioned feature can use methods described herein to provide quantitative quite accurately as the institute's cls analysis thing in the complex mixture.

Equipoise (or connector) part:

Whether take place according to the reaction with analyte, the equipoise (or connector) that can be used for the labelled reagent in the embodiment of the present invention partly (uses sometimes and is called for short " LK ") will be reported that the thing part links to each other with analyte or will report that the thing part links to each other with reaction active groups.The neutral substance (promptly experiencing neutrality in mass spectrograph loses) that can select connector in mass spectrograph, all to rupture with generation connection connector and the key (RL key) and the key that is connected connector and analyte (LA key) of report thing part.Thereby connector can be designed to when experience dissociation energy level, carry out the neutral fragment that inferior fracture only produces connector.Connector can be designed to produce one or more detectable fragments.

About isobaric labelled reagent group, connector part can comprise one or more heavy atom isotopes, so that in its mass compensation mixture every kind is labeled the rough qualitative difference of the labelled reagent of the report thing part of analyte or group and/or test kit.And, be labeled for the analyte or for the labelled reagent of group and/or test kit, the rough quality of total (promptly making the as a whole rough quality for the treatment of) of report thing/connector combination (promptly reporting thing/connector part) can be identical for every kind in the mixture.More specifically, the unique rough quality of report thing part be labeled that sample that analyte is originated is associated and the rough quality of total of described report thing/connector combination is labeled for every kind in the sample mixture under the situation of all identical (not considering the sample that sample mixture is originated) for the analyte, the connector part can compensate from the report thing that is labeled analyte of different samples (every kind of sample all utilizes with the different reagent of measuring in the dystopy group and carries out mark) partly between in rough qualitative difference.Like this, when being labeled and mixing then and when forming sample mixture, the rough quality of same analyte can have identical rough quality in two or more samples.

For example, organize and/or be used for labelled analyte test kit be labeled analyte or labelled reagent can be isobaric.Therefore, if in mass spectrograph, from the first mass analysis of sample mixture, select to have the ion (i.e. Xuan Ding ion) of specific mass-to-charge ratio (taking from sample mixture), then can represent by the described selected respective concentration and/or the amount of ion in sample mixture pro rata from the same analyte of different samples (it forms sample mixture).Therefore, connector will report that not only thing and analyte couple together, and it also can be used for compensating the mass discrepancy of unique report thing part, thereby make the rough uniform quality of the report thing/connector part that is labeled analyte in the several samples.(reaching wherein two groups of same amount dystopy labelled reagents of example) referring to Fig. 4 a

Because connector can be used as in the labelled reagent mass balance thing of report thing part, so the atomicity in the connector is big more, different isometry in group and/or the test kit/just big more with possible the number of amount dystopy labelled reagent.In other words, the atomicity that common connector is comprised is big more, the number of possible report thing/connector combination is just big more, because being substituted on almost can any position in connector, isotropic substance produces the isomers of connector part and/or with amount dystopy thing, wherein connector partly is used to offset the mass discrepancy of report thing part, thereby produces the unique isometry and/or the group of different in nature labelled reagent of poor quality.These not on the same group labelled reagent be specially adapted to the multiple analysis of analyte in the identical and/or different sample.

The sum of labelled reagent can be 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or more in group and/or the test kit.The diversity of labelled reagent is only reported atomicity in thing and the connector part, be can be used for replacing the heavy atom isotope of light isotope and the restriction of can synthesis mode settling isotopic multiple synthetic configuration in group or the test kit.Yet as noted above, the basic parent material of multiple isotopic enrichment can obtain from manufacturers (for example Cambridge Isotope Laboratories and Isotec) expediently.The basic parent material of these isotopic enrichments can be used for producing with the method for amount dystopy with different in nature labelled reagent of poor quality (labelled reagent that promptly has different rough quality), perhaps be used for producing the method for the parent material of isotopic enrichment, the parent material of described isotopic enrichment can be used for producing in groups the same amount dystopy and the synthetic method of different in nature labelled reagent of poor quality.

In some embodiments, described labelled reagent comprises the group of different in nature label of poor quality.For these groups, connector part does not randomly comprise any heavy atom isotope, because described connector is not used for " balance " different report thing usually.Like this, every kind of different reagent have unique quality and unique report thing part in described group, when the report thing part of described uniqueness when being labeled analyte and rupturing, can produce the characteristic ion of uniqueness from described in mass spectrograph.

Preparation is applicable to that some example of the labelled reagent in the labelled reagent group describes in detail below.For example, the connector part can be by formula I ^#Representative:

Wherein, X ₁, K, R ₁And R ₂The atom or the group of representative describe in detail below.

The combination (promptly reporting thing/connector part) of report thing/connector

Labelled reagent can comprise each other directly key report thing part and connector part even.As indicated above, in some embodiments, report thing/connector part all is being identical roughly for the group of labelled reagent (promptly for isobaric reagent set) and/or each member in the test kit qualitatively.And, connect the report thing and partly can be designed to when standing dissociation energy, in the ion that a part is selected, rupture at least, thereby report the thing ion from connector part and/or connector/analyte part (no matter and whether described reagent set is isobaric) release with connector key partly.Therefore, can in analyzing, directly observe MS/MS described report thing ion (in mass spectrograph, recently observing) and intensity thereof as m/z.

Report thing/connector part can comprise the multiple combination of the identical or different heavy atom isotope in the multiple labelled reagent in group or the test kit.In scientific literature, this is sometimes referred to as " coding ", " isotope-coded " or is called for short " coding ".For example, Abersold etc. discloses isotope-coded affinity tag (ICAT; Consult WO00/11208).In one aspect, the reagent of Abersold etc. is different with labelled reagents more of the present invention, and Abersold does not instruct the labelled reagent of two or more equals in quality, for example isometry and/or isobaric labelled reagent.On the contrary, Abersold etc. has instructed " gently " form and " weight " form of its labelled reagent.About of poor quality different in nature labelled reagent disclosed herein, the reagent of Abersold does not rupture in mass spectrograph and discharges fragment (son) the ionic characteristic ion that can observe analyte.Therefore, be different from the reagent of Abersold, of poor quality different in nature reagent disclosed herein allows by analyzing one MS/MS collection of illustrative plates or being suitable for forming the single data set of described collection of illustrative plates and analyte is identified and quantitatively.

In some embodiments, report thing and/or connector part can comprise and can be used for labelled reagent or be labeled analyte being fixed on atom or group on the upholder.Fixing can be direct or indirect.For example, under following situation, can take place directly fixing: atom relevant with report thing and/or connector or group (for example reporting the alkylamine substituting group of thing and/or connector) can be fixed realizing with reaction active groups (for example cleavable linker) direct interaction of upholder in some embodiments.Comparatively speaking, fix if for example report reaction active groups (for example avidin or streptavidin) interaction and realization that the substituting group (for example reporting the alkylamine substituting group of thing and/or connector) of thing and/or connector is modified (for example by biotinylation) and described modification group and upholder, indirect securement then takes place.Therefore, such embodiment is contained in the present invention: analyte can react with the labelled reagent that is combined on the upholder, and wherein every kind of upholder comprises unique labelled reagent, thus different samples and different upholder reactions; Such embodiment is also contained in the present invention: every kind of different sample and different labelled reagent reactions, and reaction product is fixed on thereafter on the identical or different upholder.In either case, all be labeled the sample mixture that analyte obtains to be used for mass spectroscopy by cutting away usually from upholder.

Mass spectrograph/mass spectroscopy (MS):

Utilizable energy is enough selected and tandom mass spectrometer and other mass spectrograph of the molion that ruptures are implemented method of the present invention.Tandom mass spectrometer can be selected and the molion that ruptures (the single-stage mass spectrograph also has this ability, but degree is lower) according to the mass-to-charge ratio (m/z) of molion, writes down fragment (son) ionic spectrum of gained then.More specifically, sub-fragment ions spectrum can produce by selected ion being applied dissociation energy (for example dissociation energy (CID) is brought out in collision).For example, can in first mass analysis, select, make its fracture and in second mass analysis, analyze again corresponding to the ion that is labeled peptide with specific m/z ratio.The representative instrument that can carry out such series connection mass analysis includes but not limited to magnetic four sectors (magnetic four-sector), series connection flight time, triple quadrupole bar, ion trap and mixing quadrupole flight time (Q-TOF) mass spectrograph.

The mass spectrograph of these types can with multiple ionizer coupling, described ionizer includes but not limited to electron spray ionisation (ESI) and substance assistant laser desorpted attached ionization (MALDI).Ionizer can be used for producing charged material, and it is used for carrying out first mass analysis when analyte does not also have fixed charge.Other mass spectrometer and fracture method comprise post-source decay in the MALDI-MS instrument and the high energy CID that uses MALDI-TOF (flight time)-TOF MS to carry out.Can consult the MassSpectrometry in Proteomics.Chem.Rev.101:269-295 (2001) of R.Aebersold and D.Goodlett about the nearest summary of tandom mass spectrometer.

The fracture of being undertaken by dissociation energy:

Generally acknowledged, key can rupture owing to the process that takes place in mass spectrograph.In addition, can be by ion being applied the fracture that dissociation energy is induced key in mass spectrograph.For example, can in mass spectrograph, produce dissociation energy by collision induced dissociation (CID).Other non-limiting example that is used in the mass spectrograph fracture ionic dissociation energy comprises that dissociate (CAD), the photoinduction of crash-active are dissociated (PID), spatial induction dissociates (SID), electronic induction dissociates (EID), electron capture dissociation (ECD), heat/black matrix ir radiation are dissociated (BIRD), post-source decay or its combination.The those of ordinary skill of field of mass spectrometry will appreciate that other example technique that applies the dissociation energy that causes fracture includes but are not limited to photodissociation, electron capture and spatial induction and dissociates.

Method by the collision induced dissociation breaking bonds comprises by selecting ionic kinetic energy state and bring up to the point that bond rupture takes place with the rare gas element collision.For example, can be by in collision cell, transmitting kinetic energy with rare gas element (for example nitrogen, helium or argon) collision.The amount that can be passed to this ionic kinetic energy is proportional with the gas molecula number that allows to enter collision cell.When having more gas molecule, can be to the selected more substantial kinetic energy of ion transport, when the gas molecule that exists more after a little while, can transmit less kinetic energy.

Therefore, clearly, the dissociation energy that can control in the mass spectrograph to be applied.Generally acknowledge that also some key is more unstable than other key.The unstable of key depends on the character of this analyte and the character of this report thing/connector/non-encoded detectable label thing part in analyte or report thing/connector/non-encoded detectable label thing part.Therefore, dissociation energy be can regulate, thereby analyte and/or labelled reagent (for example reporting thing/connector combination) fracture made in the mode that can measure.It will be understood by those skilled in the art that how mass spectrometric assembly is carried out such routine adjustment and obtains suitable dissociation energy level thus, thereby make at least a portion ion fragmentation that is labeled analyte become characteristic ion (promptly reporting the thing ion) and sub-fragment ions.

For example, can apply dissociation energy to selection/isolating ion in first mass analysis.In tandom mass spectrometer, can make the ion of extraction stand dissociation energy, thereby cause fracture, then be transferred in second mass analyzer.Selected ion can have selected mass-to-charge ratio.Described mass-to-charge ratio can be in certain mass charge ratio range, and this depends on mass spectrometric characteristic.When using collision induced dissociation, can it be transferred to second mass analyzer from first mass analyzer by making ion pass collision cell, in described collision cell, can apply dissociation energy to produce fragment ions.For example, be sent to (not fracture) selected ion (if any) that ion that second mass analyzer analyzes can comprise that some or a part are residual and report thing ion (characteristic ion) and the sub-fragment ions that is labeled analyte.

Determine analyte by the computer-Aided database analysis:

In some embodiments, can determine analyte based on the daughter ion fracture mode, described pattern is by carrying out computer assistedly relatively analyzing with spectrogram known or " theory " analyte.For example, the peptide ionic daughter ion spectrum that ruptures under low energy CID condition can be thought the summation of many discrete fracture incidents.Usual nomenclature keeps charged peptide fragment to distinguish sub-fragment ions (Reopstorff etc., Biomed.MassSpectrom., 11:601 (1988)) after according to amido linkage that is ruptured and bond rupture.Charge retention causes forming b-type ion in that the N of fracture amido linkage is distolateral.If charge retention is distolateral at the C of fracture amido linkage, then fragment ions is called y-type ion.Except b-and y-type ion, the CID mass spectrum also can contain other diagnostic fragment ions (they comprise owing to glutamine, Methionin and arginine generation ammonia neutrality lose (17amu) or the amino acid (as Serine and Threonine) that contains hydroxyl water takes place loses (ion that 18amu) produces); Described diagnostic fragment ions and b-and y-type ion all are sub-fragment ions.Observed some amino acid and under low energy CID condition, be easier to fracture than other amino acid.This is obvious especially for the peptide that contains proline(Pro) or asparagicacid residue, more obvious at aspartoyl-proline(Pro) key place (Mak, M. etc., Rapid Commun.MassSpectrom., 12:837-842 (1998)).Therefore, Z " '-pro dimer or Z " '-asp dimer (wherein Z " ' be any natural amino acid, pro is a proline(Pro), asp is an aspartic acid) peptide bond compare usually more unstable with the peptide bond in the every other amino acid dimer combination.

Therefore, with regard to peptide and protein example, low energy CID spectrum contains eclipsed b-and y-series ion, loses the sequence-specific information of the redundancy of ions from the interior segments ion of same peptide and ammonium and other neutrality.It is explain that though these CID spectrums are challenging and time-consuming from the beginning to compile parent's peptide ammino acid sequence, but still feasible.The latest developments of the computer assisted from the beginning method that is used to check order are described in Huang, Y., Ross, P, Smirnov, I, Martin, S. and Pappin, D.2003, Proceedings of 6th International Symposium on MS inHealth and Life Sciences ,-28 days on the 24th August in 2008, San Francisco CA.Most important progress was to have developed the computerized algorithm that peptide CID spectrum is associated with existing peptide sequence in protein and dna sequence data storehouse during peptide sequence was identified.The for example following program of such method: 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)).

In brief, experimental peptide CID spectrum (MS/MS spectrum) is mated or related with composing by " theory " sub-fragment ions of computer generation from the peptide sequence that derives from protein or genomic sequence data storehouse.This coupling or the related similarity that is based between the observation quality of estimating quality and MS/MS pattern neutron fragment ions.Come possible coupling or related scoring with degree of agreement between " theory " fragment is composed according to experiment.The parameter that given peptide ammino acid sequence is retrieved has differentiation power very much, to such an extent as to single peptide CID composes any given protein that is enough to identify in full genome or expressed sequence tag (EST) database.Other summaries can be consulted 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 can be used for determining to be labeled the analyte of analyte, and it also can be used for the analyte that definite institute determination and analysis thing is originated.For example, in MS/MS analyzes to the evaluation of peptide can be used for determining this peptide from which kind of protein owing to proteinic enzymic digestion cuts down.Can predict such analysis and can be used for other analyte, for example nucleic acid, lipid, steroid and/or prostaglandin(PG).

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, in the selected ion of a part, RL key and LA key can rupture when standing the dissociation energy level at least.Therefore, in some embodiments, can regulate the dissociation energy level in the mass spectrograph, make that RL key and the LA key in the selected ion of at least a portion that is labeled analyte all ruptures.

The fracture of RL key discharges described report thing part from analyte, measure described report thing ion thereby can be independent of analyte.The fracture of LA key discharges described report thing/connector part from analyte, perhaps discharge described connector from analyte, and this depends on whether the RL key ruptures.In some embodiments, the RL key can be more unstable than LA key.In some embodiments, the LA key can be more unstable than RL key.In some embodiments, RL can have identical unstable with the LA key.In brief, discharge described report thing ion thereby the RL key is designed in various embodiments of the present invention fracture, but the LA key can rupture or not rupture.

In some embodiments, when the goal analysis thing is protein or peptide, the relative instability that can regulate RL key and LA key with respect to acid amides (peptide) key.RL key, LA key or its two can be higher, identical or lower than the unstable of typical acid amides (peptide) key.For example, under the dissociation energy condition, than Z " peptide bond in '-pro dimer or Z " '-asp dimer, RL key and/or LA key can be not easy to fracture, wherein Z " ' be any natural amino acid, pro is a proline(Pro), asp is an aspartic acid.In some embodiments, key RL and key LA can rupture comparing under the roughly the same dissociation energy level with typical amido linkage.In some embodiments, RL key and key LA can rupture comparing under the higher dissociation energy level with typical amido linkage.

In some embodiments, also can there be such key RL and LA, make the fracture of key LA cause the fracture of key RL, and vice versa.Like this, key RL and key LA can rupture basically simultaneously, and making does not have the analyte (or its sub-fragment ions) of significant quantity to comprise the part marker." analyte of significant quantity " mean in mass spectrograph (for example MS/MS analyze), can detect less than 25%, preferably less than the analyte of 10% part mark.

Because there are clear and definite boundary in the labeled fragment of analyte and unmarked fragment in mass spectrum (for example MS/MS analyzes) in some embodiments, therefore this feature can make by computer-aided analysis identification of analytes from sub-fragment ions spectrum and be simplified, because the Mass Calculation that the sub-fragment ions of analyte is analyzed is not needed residual marker is compensated being used for.And, because the fragment ions of analyte can be by complete mark or unmarked (but not being the part mark) in some embodiments, therefore can seldom have or not have owing to striding across the sub-fragment ions that isotopic distribution caused between the breaking bonds, just as all having isotopic situation by each side that applies single labile bond in the caused part labelled analyte that fracture caused that is labeled analyte of dissociation energy level in qualitative distribution.

The mark of analyte:

As indicated above, the functional group that can be by making analyte and the reaction active groups of labelled reagent react labelled analyte.Functional group on the analyte can be an electrophilic group, and the functional group of labelled reagent can be a nucleophilic group.Electric thing of parent and nucleophilic thing can react and form covalently bound between analyte and labelled reagent.

Labeled reactant can take place in solution.In some embodiments, one of analyte or labelled reagent can combine with upholder.Sometimes labeled reactant can carry out under aqueous conditions.Can select aqueous conditions to be used for for example mark of protein, peptide and/or nucleic acid of biomolecules.Sometimes labeled reactant can carry out in the mixture of organic solvent or organic solvent.Can select organic solvent to be used for micromolecular analyte.The mixture of water and organic solvent or organic solvent can be extensive use of.For example, can prepare water and about 5% to the solution of about 95% organic solvent (volume/volume) and be used for labelled analyte.In some embodiments, can prepare water and about 50% to the solution of about 95% organic solvent (volume/volume) and be used for labelled analyte.In some embodiments, can prepare water and about 65% to the solution of about 80% organic solvent (volume/volume) and be used for labelled analyte.The non-limiting example of organic solvent comprises N, N '-dimethyl formamide (DMF), acetonitrile (ACN), N-crassitude (NMP) and alcohol, for example methyl alcohol, ethanol, propyl alcohol and/or butanols.Those skilled in the art can utilize the disclosure of the knowledge of this area and this paper and in conjunction with normal experiment, determine suitable solvent condition according to the character of labelled reagent and the character of analyte, so that analyte is carried out mark.

When carrying out labeled reactant, can regulate pH.PH can be 4-10.PH also can be outside this scope.Usually, can be by adding the basicity that non-nucleophilicity organic bases is regulated non-aqueous reaction.The non-limiting example of suitable alkali comprises N-methylmorpholine, triethylamine and N, the N-diisopropylethylamine.Perhaps, can use biological buffer (for example N-[2-hydroxyethyl] piperazine-N '-[2-ethanesulfonic acid) (HEPES) or 4-morpholino b acid (MES)) or inorganic buffer agent (for example yellow soda ash and/or sodium bicarbonate) regulate the pH of water-containing solvent.Because at least one described reaction active groups can be an electrophilic group, the damping fluid that selection does not comprise any nucleophilic group may be an ideal.Those skilled in the art can determine to can be used for the aignment mark reaction after carrying out normal experiment pH is beneficial to labelled reagent analyte be carried out the other buffers of mark.Therefore, those skilled in the art can be according to the character of labelled reagent and the character of analyte, only uses the disclosure of this paper and define suitable solvent condition and the pH that is beneficial to the analyte mark in conjunction with normal experiment.

Sample preparation:

In some embodiments of the present invention, can be before labelled analyte and handle sample afterwards.Processing can help the mark of analyte.Processing can help the analysis to sample composition.Processing can be simplified the operation to sample.Processing can help above-mentioned two or more multinomial.

For example, can be with enzyme or chemical treatments sample.Described enzyme can be proteolytic enzyme (with degrade proteins and peptide), nuclease (with degraded nucleic acid) or some other enzyme.Can select to have the enzyme of highly measurable degradation model.Two or more proteolytic enzyme and/or two or more ribozymes can also be used together or use with other enzyme, with the degraded sample component.

For example, protein lyase trypsinase is a kind of serine protease, the peptide bond between its cutting Methionin or arginine and the nonspecific amino acid, thus produce the peptide that comprises aminoterminal (N end) and Methionin or arginine carboxyl terminal amino acid (C end).Like this, the peptide that the protein cutting is produced is foreseeable, and its exist situation and/or amount in tryptic digestion product sample can be indicated its proteic situation and/or amount of existing in source.In addition, the unhindered amina end of peptide can be the good nucleophilic thing that helps its mark.Other exemplary protein lyases comprise papoid, stomach en-, ArgC, LysC, V8 proteolytic enzyme, AspN, PRONASE A, Chymotrypsin and carboxypeptidase (for example Carboxypeptidase A, B, C etc.).

For example, protein (for example protein g) can produce three kinds of peptides (for example peptide B, C and D) using when digesting such as tryptic proteolytic enzyme.Therefore, used protein lyase (as trypsinase) to digest and the sample that in analysis, confirms to contain peptide B, C and D we can say comprise at first as described in protein g.The amount of peptide B, C and D is also relevant with the amount of protein g in the institute sample digestion.Like this, any evaluation and/or quantitatively in the sample (or its fragment) mensuration of one or more among peptide B, C and the D all can be used for protein g in the primary sample (or its fragment) is identified and/or quantitatively.

Because the activity of enzyme is predictable, therefore the peptide sequence that produces from the degraded of sequence known protein matter is foreseeable.Use this information can produce " theory " peptide information.Therefore, determine that in the computer-aided analysis that the sub-fragment ions (as mentioned above) of actual sample mass spectroscopy is carried out " theory " peptide fragment can be used for one or more peptides or the protein in definite one or more unknown samples.(being the part of " determining analyte ") by the computer-Aided database analysis referring to for example above-mentioned title.

In some embodiments, sample preparation can comprise the precursor of handling analyte to be marked.For example, the peptide of digesting protein and selective marker reagent (for this example) make it and the reaction of this peptide or peptide analysis thing if analyte to be marked is hung oneself, and then can handle protein (analyte precursor molecule) in the sample in the mode that helps labeled reactant.In this example, can go back crude protein with reductive agent (for example three [2-propyloic] phosphuret-(t)ed hydrogen (TCEP)), then by with closed reagent (for example methyl thiosulfonic acid methyl esters (methyl methanethiosulfonate, reaction MMTS) and sealing.Like this, proteinic thiol group is closed, thus the amine of not interferometric analysis thing and the labeled reactant between the labelled reagent.

Skilled person in the art will appreciate that and to use the reagent and the adjustable scheme that are easy to obtain also can be undertaken by normal experiment to the processing of some other precursor molecule.The definite selection of reagent and condition can be carried out 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 support no matter whether with labelled reagent this solid support has been carried out 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 reducing analyte.In some embodiments, the fixing mark that can help analyte.In some embodiments, the fixing mark that can help the analyte precursor.In some embodiments, fixingly can help the fraction in the sample component that comprises some characteristic (for example it comprises or lack the halfcystine part) is carried out selected marker.In some embodiments, fixingly can help purifying.Fixing can help above two or more multinomial.

Separate, comprise the sample separation mixture:

In some embodiments, can comprise separation to the sample that is labeled analyte or the processing of sample mixture.Can carry out one or many to mark or unlabelled analyte, mark or unlabelled analyte precursor or its fraction separates.Can carry out one or many to other product of one or more fractions that derive from solid-phase capture or sepn process separates.Can separate two or more aforementioned sample types, and can before mass analysis, carry out dissimilar separation.

For example, can prepare the sample mixture that comprises from the otherness labelled analyte of different samples.The otherness mark means every kind of marker and comprises appraisable unique property.(the report thing part that for example in MS/MS analyzes, comprises the uniqueness that produces unique " characteristic ion ").For the analytic sample mixture, can separate the component of this sample mixture, and only a kind of fraction of this sample mixture be carried out mass analysis.Like this, can significantly reduce the complicacy of analysis,, thereby improve Sensitivity of Analytical Method because can analyze quality independently to isolating analyte.Certainly, can repeat the one or many analysis, thereby allow all fractions in the sample mixture are analyzed one or more other fractions of sample mixture.

In every kind of sample that can use following separation condition to measure to comprise sample mixture every kind is labeled the amount (prerequisite is that the amount that joins every kind of sample in the described sample mixture is known) of analyte: under the described separation condition by the same analyte of otherness mark under certain concentration or with certain amount by co-elute, described concentration or amount and its abundance in sample mixture are proportional.Therefore, in some embodiments, the separation of sample mixture can make to analyze to be simplified, and keeps simultaneously in middle institute's measured signal of mass analysis (for example MS/MS analyzes) and the sample mixture by the dependency between the amount of the analyte of otherness mark.

Separation can be undertaken by chromatography.For example, can use liquid chromatography/mass spectrometry method (LC/MS) to realize sample separation and mass analysis.In addition, any chromatography separating method that is suitable for separating the goal analysis thing can use.For example, chromatographic separation can be normal phase chromatography, reverse-phase chromatography, ion exchange chromatography (being anion-exchange chromatography or cation-exchange chromatography), molecular size exclusion chromatography or avidity chromatography.

Separation can be undertaken by 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.

Isobaric labelled reagent or reagent set can be used for the analyte of mark sample, when carrying out separating step, isobaric labelled reagent is particularly useful, because the isobaric marker in the labelled reagent group is undistinguishable basically (and can not distinguish by remove the segmental rough quality of report behind the thing from described analyte).Therefore, mode (for example co-elute) that can be identical just by all analytes of the different dystopy of amount together marker marks in the same composition is carried out chromatographic separation.Because they are undistinguishable basically, so can comprise the analyte of a certain amount of every kind of quilt with amount dystopy mark from the elutriant of sepn process, the amount that is labeled analyte in described analyte and the sample mixture is proportional.In addition, according to for the understanding that how to prepare sample mixture (adding the sample of each several part and other optional component (for example calibration standard thing)) with the preparation sample mixture, the amount that is labeled analyte in the sample mixture can be returned be labeled sample that analyte is originated in the analyte that is labeled be associated.

Described labelled reagent can also be different in nature labelled reagent of poor quality (labelled reagent that promptly has different rough quality).Different in nature labelled reagent of poor quality can be used to for example analyte in two or more different samples of mark, wherein in the group different labelled reagents all have different quality (promptly with group in other reagent compare known mass discrepancy).Because all reagent all can have known mass discrepancy in the group, so the relative quantity that need not make the labelled reagent fracture come similar analysis thing in quantitative two kinds of different samples.Yet in some embodiments, described labelled reagent can rupture.

Relative and the absolute quantitation of analyte:

In some embodiments, might carry out relative quantification to the same analyte of otherness mark in the sample mixture.For example, might come the same analyte of otherness mark is carried out relative quantification by the relative quantity (for example peak area of being reported and/or height) of the report thing ion (being characteristic ion) relatively in mass analysis (observe in for example to first mass analysis selected be labeled second mass analysis that analyte carries out), measured.In other words, when every kind of report thing ion can be associated with the information of the every kind of specific sample that is used to produce sample mixture, then this report thing ion was exactly the relative quantity of this analyte in sample mixture with respect to other report thing ionic relative quantity that is observed in the mass analysis.When the component of forming sample mixture known (amount of every kind of sample of for example forming sample mixture is known), then can be based on the report thing ionic relative quantity that analyte observes of being labeled with selected mass-to-charge ratio is calculated the relative quantity of this analyte at the every kind of sample that is used for preparing this sample mixture conversely.Can repeat this process to all differences labelled analyte that observes in first mass analysis.Like this, can measure the relative quantity (often showing) of the every kind of reactive behavior analyte of every kind of different samples that is used for producing sample mixture with concentration and/or scale.

In other embodiments, can determine the absolute magnitude of analyte.With regard to these embodiments, can in sample mixture, add one or more otherness labelled analytes (calibration standard thing) of known quantity, perhaps report thing ionic intensity can be associated with calibration curve.

The calibration standard thing can be with the isometry in the marker group that is used for mark sample mixture analyte and/or with measuring the predictive analysis thing that the dystopy marker has carried out mark, all is unique as long as the report thing of this calibration standard thing part is compared with any sample that forms this sample mixture.In case determined the report thing ionic relative quantity of the report thing ion of calibration standard thing, just might calculate the absolute magnitude (often showing) of all differences labelled analyte in this sample mixture with reference to the amount that adds the calibration standard thing in this sample mixture with concentration and/or scale with respect to otherness labelled analyte in the sample mixture.Like this, can also determine every species diversity labelled analyte absolute magnitude of (in the sample in this analyte source, having the calibration standard thing) based on the knowledge of this sample mixture of preparation.

Perhaps, can produce calibration curve by the representative sample that analysis is labeled analyte, wherein every kind of sample comprises the analyte that is labeled of different known quantities.The report thing ionic peak intensity that is labeled analyte analyzed (with amount dystopy group) or the peak intensity (opposite sex group of poor quality) that is labeled analyte can be labeled the analyte mapping to every kind of known quantity, thereby produce typical curve.In case the generation typical curve just can compare report thing ion in the unknown sample or the intensity and the typical curve that are labeled analyte (if desired), thereby the amount of analyte in definite specimen.

Though foregoing is arranged, can also just report if desired in the thing part that any natural existence or the artificial isotopic abundance that produces proofread and correct the intensity of report thing ion (characteristic ion).There is multiple mode to proofread and correct the isotopic abundance of impurity in the characteristic ion of report thing part.A this gauged example is found in the common unsettled total U.S. Provisional Patent Application No.US 2005-0114042A1 that the disclosed name of submitting on August 12nd, 2004 is called " Method and Apparatus For De-Convoluting A Convoluted Spectrum ".Basically, can use mathematical formula and calculating, measure the intensity at upper limit quality (up-mass) relevant and lower limit quality (down mass) peak by the convolution spectrum of the overlapping isotope group of labelled reagent is deconvoluted (deconvolution) with isotope group in the single marking reagent.No matter value is how to record, the intensity of quantitative exactly more every kind of report thing ion (being characteristic ion), and the relative and absolute quantitation of analyte is just accurate more in the primary sample.

Proteomic analysis:

Embodiment of the present invention can be used for complex analyses, because can utilize the mass analysis technology with fast and the multiple mode is compound with sample, analyze and analysis again.For example, can analyze the amount of one or more analytes in one or more samples.Can analyze the amount (often showing) of analyte in the sample of forming sample mixture with concentration and/or scale.Because sample preparation and mass analysis can be carried out fast, therefore these methods can repeat repeatedly, make many otherness labelled analytes in the sample mixture amount can at its in the sample that analyte is originated relatively and/or absolute magnitude measure.

Can use an application of this quick multiple analysis is the Proteomic analysis field.Proteomics can be regarded the experimental method of the coded information in the genome sequence of describing as aspect structure, function and bioprocess adjusting.This can or organize expressed holoprotein group component to carry out systematicness analysis and realize by pair cell.With the mass spectroscopy of method of the present invention, mixture, test kit and/or the coupling of composition embodiment be a kind of possible instrument of this overall protein analysis.

For example, for one group 9 kinds same amount dystopy labelled reagents, in experiment, can obtain 9 time points to measure the rise or the downward modulation of protein expression.For example, based on cell the replying in the growth to particular stimulation.Can also carry out less time point but introduce one or more contrasts.Can also in same multiple experiment, carry out dual mensuration or triplicate.In all cases, all can in the multiple experiment of single, measure the rise or the downward modulation of protein expression, randomly exist under one or more optional contrasts and/or sample carries out under repeating.In addition, because be parallel carrying out,, thereby need not compensate the subtle change of interpretation scheme or experiment condition so the result is directly comparable to the processing of sample mixture.Therefore, can use these experimental analyses to include but not limited to time course experiment, biomarker analysis, multiplexed protein matter group analysis, the experiment of multidimensional protein identification techniques, affinity capture, posttranslational modification (PTM) mensuration and multiple control experiment with amount dystopy labelled reagent.

The II composition

In some embodiments, the present invention relates to the composition of formula I (comprising its salt form and/or hydrate forms) representative:

Wherein group Y-J can be any report thing group.The character of suitable report thing group was described at preamble.The character of suitable report thing group also is described among the U.S. publication application No.US2004-0219685-A1, especially the 41-47 section.

For example, the report thing can comprise 5,6 or 7 yuan of heterocycles as group Y, wherein said heterocycle can be replacement or unsubstituted, and can randomly can link to each other with upholder with cutting, and wherein said heterocycle comprises at least one theheterocyclic nitrogen atom that links to each other with group J by covalent linkage.Group J replaces or unsubstituted methylene group, by formula-CJ ' ₂-expression, 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 ₂-(CK ' ₂) _m) _pThe group of-representative, wherein n is 0 or 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 ₄About radicals R ₁And R ₂: (1) R ₁Be hydrogen, deuterium or R ₆, R ₂Be hydrogen, deuterium or R ₇Perhaps (2) R ₁And R ₂Be together by formula-(CR ' ₂) _q-or-((CR ' ₂) _m-X ₂-(CR ' ₂) _m) _pThe group of-representative, it forms the ring of described two nitrogen-atoms of bridge joint, wherein q is 1～10 integer, each m is 1～5 integer independently of one another, p is 1～4 integer, each R ' be independently of one another hydrogen, deuterium, fluorine, chlorine, bromine, iodine ,-R ₅,-OR ₅,-SR ₅,-R ₅' OR ₅Or-R ₅' SR ₅Atom or radicals X ₁Can be=O ,=S ,=NH or=NR ₇Atom or radicals X ₂Can be-O-or-S-.Group Z can be hydrogen or covalently bound analyte.Each R ₃, R ₄, R ₅, R ₆And/or R ₇Can be alkyl, thiazolinyl, alkynyl, aryl, heteroaryl or arylalkyl independently of one another.For example, each R ₃, R ₄, R ₅, R ₆And/or R ₇Can be methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, isobutyl-, sec-butyl or the tertiary butyl independently of one another.Each R ₃', R ₄' and/or R ₅' can be alkylidene group, alkenylene, alkynylene, arylidene or alkyl arylene independently of one another.Each R for example ₃', R ₄' and/or R ₅' can be methylene radical, ethylidene, propylidene, cyclopropylidene, inferior normal-butyl, inferior cyclobutyl, inferior n-pentyl, cyclopentylidene, inferior n-hexyl or cyclohexylidene independently of one another.

In some embodiments, Y-J can be by above-mentioned formula X ¹, X ², X ³, X ⁴, X ⁵, X ⁶, X ⁷, X ⁸Or X ⁹The group of representative.In some embodiments, Y-J can be by above-mentioned formula X ¹⁰, X ¹¹, X ¹², X ¹³, X ¹⁴, X ¹⁵, X ¹⁶, X ¹⁷, X ¹⁸, X ¹⁹, X ²⁰, X ²¹, X ²², X ²³, X ²⁴, X ²⁵Or X ²⁶The group of representative.

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

Described 5,6 or 7 yuan of heterocycles can be comprise at least one can with any 5,6 or 7 yuan of heterocycles of the covalently bound nitrogen-atoms of group J.For example, it can be to replace or unsubstituted morpholine, piperidines or piperazine.Possible substituting group has been described in above-mentioned " definition " part, 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 (' D ' is deuterium in each case).Described substituting group can link to each other with the heteroatoms of ring.For example, described heterocycle can be a N methyl piperazine.Described heterocycle can be aromatics or non-aromatics.

In some embodiments, report thing part can link to each other with upholder with cutting.Multiple upholder is well-known in the art.For example, the various upholders that comprise trityl part are commercial on sale or can make (for example trityl chloride upholder (Trityl-Cl) or 2-chlorine trityl chloride upholder).

For example, the hydroxyl of the report thing of labelled reagent part or thiol group can react with the cleavable linker of suitable upholder.Described cleavable linker can be " a sterically hindered cleavable linker ".The cutting of cleavable linker will discharge marker or be labeled analyte from upholder.The non-limiting example of sterically hindered solid support comprises: trityl chloride resin (trityl-Cl, Novabiochem, P/N 01-64-0074), 2-chlorine trityl chloride resin (Novabiochem, P/N 01-64-0021), DHPP (Bachem, P/N Q-1755), MBHA (Applied Biosystems P/N 400377), 4-methyl trityl chloride resin (Novabiochem, P/N 01-64-0075), 4-methoxyl group trityl chloride resin (Novabiochem, P/N 01-64-0076), hydroxyl-(2-chloro-phenyl-) methyl-PS (Hydroxy-(2-chorophnyl) methyl-PS) (Novabiochem, P/N01-64-0345), Rink acid resin (Novabiochem P/Ns 01-64-0380,01-64-0202), NovaSyn TGT alcohol resin (Novabiochem, P/N 01-64-0074).Multiple other cleavable linker is as known in the art, can utilize these cleavable linkers and only use commercially available material, normal experiment and this paper the instruction that is provided to prepare suitable upholder.

Therefore, in some embodiments, described 5,6 or 7 yuan of heterocycles can include and are beneficial to it and carry out cutting atom or the group that is connected with suitable upholder.For example, described group can be alkylidene group, alkenylene, alkynylene, arylidene or the alkyl arylene that contains amino, hydroxyl or thiol group.Described atom can be the secondary nitrogen of piperazine ring.Argumentation for exemplary diethylenediamine compound and its preparation method is found among the laid-open U.S. Patents application No.US 2004-0219685A1.For example, the N-alkylpiperazine acetic acid compound that described upholder engages can with diamine reactant, then with diacid reactant, thereby form the compound that the upholder that can be used as labelled reagent engages, wherein isotope-coded can be based on the character of reactant.

With reference to formula I, group Y-J-(no matter whether can be connected with upholder) can form report thing part with cutting again.Described report thing part can comprise the site of at least one isotopic enrichment.Described report thing part can comprise the site of at least two isotopic enrichments.Described report thing part can comprise at least 3,4,5,6,7,8,9,10,11,12,13,14,15,16 or 17 or the site of more a plurality of isotopic enrichments.

Described report thing part can comprise fixed charge or ionogenic in mass spectrograph.For example, the compound that contains basic group (for example amino) is protonated easily and introduce electric charge, acidic cpd (for example hydroxy-acid group) takes off proton easily and introduces electric charge (referring to Roth, Kennethet al, " Charge Derivatization of Peptides for Analysis by MassSpectrometry ", Mass Spectrometry Reviews, 17:255-274 (1998)).

Equipoise (connector) part can be by formula I ^#The group of representative forms:

R wherein ₁, R ₂, X ₁With K as defined above.Described equipoise part can comprise at least one isotopic enrichment site.Described equipoise part can comprise at least two isotopic enrichment sites.Described equipoise part can comprise at least 3,4,5,6,7,8,9,10,11,12,13,14,15,16 or 17 or more a plurality of isotopic enrichments site.

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

Wherein W is atom or the group that is used for replacing at least one the M group on described 6 yuan of heterocycles, and be in nitrogen on the described six-ring the neighbour, or contraposition.Group W can be-N (H)-,-N (R ")-,-N (R " ')-,-P (R ")-,-P (R " ')-,-O-or-S-.If be chosen as-N (R " ')-or-P (R " ')-, then this group can be used for making described composition can be connected with upholder with cutting.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, X ₁, R ₁, R ₂With Z as defined above.Each R " can be alkyl, thiazolinyl, alkynyl, aryl, heteroaryl or arylalkyl independently of one another, each R " ' can be H independently of one another ₂N-R ₉'-, H (R ₁₀) N-R ₉'-, (R ₁₀) ₂N-R ₉'-, HO-R ₉'-, HS-R ₉The cleavable linker that '-maybe can be connected this compound and upholder with cutting.Each R ₈And/or R ₁₀Can be alkyl, thiazolinyl, alkynyl, aryl, heteroaryl or arylalkyl independently of one another, each R ₈' and/or R ₉' can be alkylidene group, alkenylene, alkynylene, arylidene or alkyl arylene independently of one another.

In some embodiments, described composition can be by formula III (comprising its salt form and/or hydrate forms) representative:

Wherein s can be 0～5 integer.Radicals R ₁, R ₂With Z as defined above.Atom or radicals R ₁₁Can be hydrogen, deuterium, methyl ,-C (H) ₂D ,-C (H) D ₂,-CD ₃Or other alkyl or-R " ', wherein R " ' as defined above.For example, described composition one of can be selected among compound as follows (comprising its salt form and/or hydrate forms) V-XII or the XXV to XXXII:

Wherein, ^*Indicate and comprise when suitable ¹³C substitutes ¹²C, ¹⁵N substitutes ¹⁴N or ¹⁸O substitutes ¹⁶The isotopic enrichment site of O, Z is hydrogen or covalently bound analyte.

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

Wherein, R ₁₁With Z as defined above.For example, described composition optional shown in following compound (comprising its salt form and/or hydrate forms) XV to XXIII or XXXV to XXXXI in one of:

Wherein, ^*Indicate and comprise when suitable ¹³C substitutes ¹²C, ¹⁵N substitutes ¹⁴N or ¹⁸O substitutes ¹⁶The isotopic enrichment site of O, Z is hydrogen or covalently bound analyte.

In some embodiments, described composition can be by formula III ^*(comprising its salt form and/or hydrate forms) representative:

Wherein, R ₁₁With Z as defined above.For example, described composition optional shown in following compound (comprising its salt form and/or hydrate forms) M to MV or MVI to MXIII in one of:

Wherein, ^*Indicate and comprise when suitable ¹³C substitutes ¹²C, ¹⁵N substitutes ¹⁴N or ¹⁸O substitutes ¹⁶The isotopic enrichment site of O, Z is hydrogen or covalently bound analyte.

As above-mentioned, described composition can salt form and/or hydrate forms existence.Said composition whether with salt form exist depend on usually substituent character and number with and exist and/or isolating condition.As everyone knows, can be by making basic group (as amine) protonated with acid treatment, thus form the salt of this amine.For example, the piperazine that contains labelled reagent can be used as single tfa salt, single HCl salt, two tfa salts or two HCl salt and obtains (consulting as U.S. Patent Publication US2005-0148771A1).Also well-known, can form carboxylate salt by make acidic-group (such as carboxylic acid) take off proton with alkaline purification, reference is the same.Also well-known, the compound that contains basic group (such as amine) and acidic-group (such as carboxylic acid) can exist with zwitterionic form.All these thinks salt form, and the ionization state of these functional groups depends on the pH of its existing any solution in the composition, perhaps if isolating, then depends on the pH of its separation solution.Those of ordinary skills extremely understand the disclosure of how only utilizing normal experiment and this paper the charge state and the character of any gegenion salt form of composition described herein are operated.

Whether composition exists with hydrate forms and also can be depending on it and exist or isolating condition.Hydrate only comprises the water molecules of one or more complexings.The disclosure of invention contains any possible hydrate forms or its combination.

As previously mentioned, group Z can be covalently bound with analyte.Described analyte can make by the reaction of analyte and labelled reagent.Described analyte can be any analyte.For example, group Z can be peptide or protein.

Group Z can also be hydrogen and therefore comprise the nucleophilic reaction active group.

Labelled reagent can be organized in groups, described group can be the group with amount dystopy and/or different in nature mark of poor quality.Other character of 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 isotopic enrichment (i.e. a coding).Described labelled reagent can be isotopic enrichment to comprise one or more heavy atom isotopes.Described labelled reagent can be isotopic enrichment to comprise two or more heavy atom isotopes.Described labelled reagent can be isotopic enrichment to comprise three kinds or more kinds of heavy atom isotope.Described labelled reagent can be isotopic enrichment to comprise four kinds or more kinds of heavy atom isotope.

In some embodiments, composition of the present invention can be the calibration standard thing that is labeled.As described herein, the calibration standard thing of known quantity can be joined in the mixture to assist the absolute quantitation analysis of goal analysis thing.Therefore, in some embodiments, the present invention relates to utilize analyte, such as interested peptide with amount dystopy and/or different in nature labelled reagent mark of poor quality.Therefore, the described calibration standard thing that is labeled can be any analyte that utilizes labelled reagent mark as described herein to cross.Described labelled reagent can be selected from the group with amount dystopy and/or different in nature labelled reagent of poor quality.

III. mark and analytical procedure

According to embodiments more of the present invention, the analyte mark can be measured then.Can measure by mass analysis and be labeled the one or more fragments of analyte, analyte itself, analyte and/or the fragment of marker.In some embodiments, method of the present invention can be used for analyzing the different analytes in the same sample, and is used for the identical and/or different analyte of two or more different samples is carried out multiple analysis.Can mix two or more different samples to form sample mixture.In multiple analysis, can determine analyte derives from which kind of sample in the sample mixture by applying marking reagent.Can measure this analyte absolute in every kind of described two or more samples (its combination and form sample mixture) and/or (for example with respect to the same analyte in the different samples) amount (often showing) relatively with concentration or scale.In addition, the precursor of this analyte and/or this analyte is identified in mass analysis that can operational analysis thing fragment (for example sub-fragment ions), for example under the situation that this analyte is formed by the precursor molecule degraded.

The sample that uses in this analysis can be any sample that comprises analyte, described analyte can with labelled reagent disclosed herein (such as the front under title " composition " item described those) carry out mark.For example, sample can be rough or treated cell lysate, body fluid, tissue extract or cell extract.Sample can be the fraction of sepn process.Other possible sample type was discussed 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 discussed in this article.

A difference of described method is the following fact: the analyte from different samples can carry out otherness mark (i.e. coding) with the marker of uniqueness, the marker of described uniqueness chemically is being isobaric (having same rough quality) or different in nature label of poor quality, and identifies the sample that described analyte is originated.For isobaric labelled reagent, the analyte of described otherness mark is not distinguished under mass spectrometric MS pattern, because it has same (always) mass-to-charge ratio.Often the labelled reagent in the group is selected, thereby made the described analyte that is labeled also can not pass through isolation technique (, can before first mass analysis, use it for mixture) and distinguish such as chromatography or electrophoretic method.Yet when being applied in dissociation energy (such as by collision induced dissociation (CID)), described marker can rupture and produce and can pass through the uniqueness that quality (mass-to-charge ratio) resolves report the thing ion in mass spectrograph.Can be with at MS/MS (or MS ⁿ, wherein n is the integer greater than 1) and the relative quantity that is labeled analyte in every kind of unique report thing ionic relative quantity being observed in the mass spectrum and the sample mixture is associated, and is associated by inferring with the relative quantity of this analyte in the sample of originating.Therefore, the relative intensity of report thing ion (being characteristic ion) can be used to measure the relative quantity of analyte in two or more different samples (its combination and form sample mixture).According to report thing ionic information, if mix in sample mixture with known quantity the calibration standard thing of every kind of analyte (its expectation is obtained absolute quantitation) or under the situation of the calibration curve that can obtain to report the thing ion or be labeled analyte, can draw the absolute magnitude (being often expressed as concentration and/or amount) of analyte in two or more samples.

For example, described analyte can be to utilize enzymic digestion reaction treatment sample and the peptide that obtained by protein degradation.Protein degradation can be realized by utilizing one or more proteolytic ferments (for example trypsinase, papoid, stomach en-, ArgC, LysC, V8 proteolytic enzyme, AspN, PRONASE A, Chymotrypsin or carboxypeptidase) to handle sample.By the peptide in the sample mixture being carried out qualitative and quantitative assay and identifies the sample (randomly mensuration simultaneously) that peptide is originated from other peptide in this sample, the sample that can originate with respect to peptide and described degraded propeptide albumen identified and/or quantitatively.Because this method allows one or more protein that surpass in a kind of sample (promptly from sample mixture) are carried out multiple assay, so it is a kind of multiple method.

Therefore, in some embodiments, the invention still further relates to the method that may further comprise the steps: make the different labelled reagents reactions in two or more samples (every kind of sample comprises one or more reactive behavior analytes) and the labelled reagent group, thereby produce the sample of two or more otherness marks, each self-contained one or more of described sample are labeled analyte.Described labelled reagent can be selected from the group with amount dystopy and/or different in nature labelled reagent of poor quality.

For example, the different labelled reagents in the group can be represented by formula I ' (comprising its salt form and/or hydrate forms):

Wherein atom or group Y, J, K, R ₁, R ₂And X ₁As defined above.In some embodiments, Y replaces or unsubstituted morpholine, piperidines or piperazine part.

In some embodiments, labelled reagent in the group can be with amount dystopy and/or isomeric, every kind of different labelled reagent all has identical rough quality in wherein said group, but wherein the group Y-J of every kind of different labelled reagent (described group forms report thing part) in one or more isotopic enrichments site by unique code, thereby when the group J of described group Y-J and the key between the segmental rest part of described labelled reagent rupture, produce report thing ion in mass spectrograph with unique qualities.In some embodiments, report thing part can comprise replacement or unsubstituted piperidines, piperazine or morpholine group.

In some embodiments, described labelled reagent can be one group of different in nature label of poor quality, and wherein all markers in the group all comprise different quality.Can design these labelled reagents and make it stand dissociation energy and rupture, but and nonessential design like this because analyze usually with MS ¹Pattern is carried out.Therefore, in some embodiments, the group Y-J of every kind of different labelled reagents can be encoded uniquely in one or more isotopic enrichments site in the group, when the key between the group J that makes at group Y-J and the rest part of labelled reagent ruptures, produce report thing ion with unique qualities in mass spectrograph.

No matter whether described group be isobaric (and/or isomeric) or the opposite sex of poor quality,, then labelled reagent can be selected in some embodiments so that the quality of each self-contained uniqueness if labelled reagent can produce report thing ion.Therefore, every kind of report thing ion with unique qualities can be used for identifying every kind and is labeled the sample that analyte is originated.

The reagent of formula I ' comprises the nucleophilic nitrogen that can form acid amides with the carboxylic moiety reaction of analyte.Described nucleophilic nitrogen also can react with the aldehydes or ketones of analyte, forms stable adducts but described schiff bases (shifts base) is reduced usually.Form howsoever, the analyte that is labeled of sample mixture can be by formula I " (comprising its salt form and/or hydrate forms) representative:

Wherein, atom or group Y, J, K, R ₁, R ₂And X ₁As defined above, group Z " can be covalently bound analyte.In some embodiments, variable Y can be to replace or unsubstituted morpholine, piperidines or piperazine group.

Described marking method can produce the sample of two or more otherness marks, and every kind of sample comprises one or more and is labeled analyte.In case utilize labelled reagent unique for this sample that the analyte in every kind of sample is carried out mark, then can the sample of two or more otherness marks or its is partially mixed and form sample mixture.Described sample mixture can randomly comprise one or more calibration standard things.

Can write down the volume and/or the amount of every kind of sample (its combination and form sample mixture).For the gross sample volume and/or amount of sample mixture, the volume of every kind of sample and/or amount can be used to measure the amount (often representing with concentration and/or amount) of institute's identification of analytes in every kind of sample analyzing from sample mixture.Therefore, sample mixture can comprise complicated mixture, wherein by the amount of analyte in every kind of two or more samples is carried out relative quantification, perhaps add under the situation in the sample mixture and carry out absolute quantitation will proofreading and correct standard substance, and the relative quantity of identical and/or different analytes can be identified and/or quantitatively.

For the same amount dystopy group of labelled reagent, can wherein can utilize first mass analyzer to carry out first mass analysis to mixture application of spectral technology for example to sample mixture or its fraction.Can select ion then with specific mass-to-charge ratio from first mass analysis.Apply dissociation energy (for example collision induced dissociation (CID)) for selected ion thus induce selected ionic fracture.By applying dissociation energy for the selected ion that is labeled analyte, key can rupture in the selected ion of a part at least.In some embodiments, the fracture of two kinds of keys all can cause the fracture of report thing/connector part and cause from analyte and discharges Ionized report thing part (promptly reporting thing ion or characteristic ion).The example of this fracture of multiple labelled reagent is shown among Fig. 1 a to 1c.Dissociation energy also can produce the sub-fragment ions of analyte to selected ionic fracture.Ion (remaining selected ion, sub-fragment ions and Ionized report thing part (being characteristic ion)) or its part can be guided in second mass analyzer then.

In second mass analyzer, can carry out second mass analysis to selected ion and part thereof.Described second mass analysis can be determined at least a every kind of unique rough quality of thing ionic (or m/z) and the relative quantity reported with selected mass-to-charge ratio and (rough and/or absolute) quality existence that is labeled some or all sub-fragment ions of analyte in the sample mixture.For the every kind of analyte that exists with selected mass-to-charge ratio, can utilize sub-fragment ions to identify analyte with selected mass-to-charge ratio.For example, this analysis can be as the front title for described in the part of " analyte determination that is undertaken by Computer-aided Analysis of Data ".Therefore, in some embodiments, described method also comprises the rough quality and/or the absolute mass of the sub-fragment ions of some or all in the rough quality of every kind of characteristic ion in second mass analysis and the relative quantity and second mass analysis.No matter described reagent is from amount dystopy group, opposite sex group of poor quality or with the two the combination of amount dystopy and different in nature reagent of poor quality, all might be used to produce the single quality collection of illustrative plates of quality collection of illustrative plates or the individual data group obtains for the evaluation of analyte and quantitatively, because the relevant information of thing ion and sub-fragment ions of reporting is present in the same data set or collection of illustrative plates by analysis.In some embodiments, described method also comprises by analyzing sub-fragment ions and measures relevant with the selected mass-to-charge ratio analyte (and/or its precursor) that is labeled.

In some embodiments, some step of described method can repeat one or many.For example, in some embodiments, can carry out dissociation energy to ion and handle, thereby form Ionized report thing part (being characteristic ion) and the sub-fragment ions of at least some aforementioned selected ionic from the selected mass-to-charge ratio of having of first mass spectroscopy (being different from any selected mass-to-charge ratio in the past).Can carry out second mass analysis to selected ion, report thing ion and/or sub-fragment ions or its part.Can also measure the rough quality of characteristic ion of every kind of uniqueness in second mass analysis and (rough or absolute) quality of relative quantity and sub-fragment ions.Randomly, can measure relevant with the selected mass-to-charge ratio analyte (or precursor molecule) that is labeled by analyzing sub-fragment ions.Like this, just these information can be used for evaluation that one or more other analytes from first mass analysis are carried out and/or quantitatively.

In some embodiments, sample mixture by the classification situation of (for example separating) by chromatography or electrophoretic method under, it may be useful repeating described method one or many.For example, repeat described method, might analyze whole sample mixtures by one or more other fractions at sample.Can estimate, in some embodiments, can repeat the complete method one or many, and in these repeat each time in, all can as mentioned above some step be repeated one or many.Like this, can on the maximum possible degree, seek the also content of working sample mixture.Also can be and repeat described complete method at new group of two or more samples.

The those of ordinary skill of field of mass spectrometry will appreciate that described first and second mass analyses can be carried out in tandom mass spectrometer.The instrument of mass analysis of being suitable for connecting was described in this article.Though preferred tandom mass spectrometer also can be used the single-stage mass spectrograph.For example, can rupture by awl voltage and induce the analyte fracture, then by the gained fragment being utilized single-stage quadrupole or time-of-flight mass spectrometer carry out mass analysis.In other examples, can use laser source and analyte be applied dissociation energy and write down the gained fragment, in flight time or series connection flight time (TOF-TOF) mass spectrograph, carry out post-source decay then.

As previously mentioned, in some embodiments, described labelled reagent or labelled reagent group can be that upholder engages.Therefore, be labeled the analyte except adapting to from the upholder recovery, the reagent that can utilize upholder to engage is implemented aforesaid method.In some embodiments, the present invention relates to implement above-mentioned any method, wherein every kind of different labelled reagent is that upholder engages and is connected with upholder by cleavable linker in the group, thereby every kind of different sample all can with have group in the upholder reaction of different labelled reagents, and wherein said method also is included in after the step of carrying out the mark sample but is mixing the described sample mixture for preparing before being labeled sample: i) randomly clean the composition of every kind of upholder with the nonreactive every kind of sample of reaction active groups of removing the labelled reagent that engages with upholder; Thereby ii) cutting described cleavable linker discharges from every kind of upholder and is labeled analyte, the sample of every species diversity mark comprises one or more and is labeled analyte, and wherein relevant with the specific sample analyte that is labeled is can unique qualities report thing is partly identified and/or quantitative by coupled having; And the analyte that is labeled of iii) randomly before mixing, collecting every kind of sample.

In some embodiments, method of the present invention also comprises and utilizes every kind of sample of at least a enzymic digestion with the sample composition of partly or entirely degrading before carrying out mark at the analyte to sample (also being the aforementioned part of " sample preparation " referring to title).For example, described enzyme can be proteolytic enzyme (with degrade proteins and/or peptide) or nuclease (with degraded nucleic acid).Two or more enzymes can also be used together, thereby further degrade sample composition.For example, described enzyme can be a proteolytic ferment, such as trypsinase, papoid, stomach en-, ArgC, LysC, V8 proteolytic enzyme, AspN, PRONASE A, Chymotrypsin or carboxypeptidase (for example A, B, C etc.).

In some embodiments, method also can be included in and carry out described first mass analysis sample separation mixture (also referring to the part that with heading is " separate, comprise the separation of sample mixture ") before.Like this, can only carry out described first mass analysis at the fraction of sample mixture.Separation can be undertaken by any separation method, comprises by chromatography and/or passes through electrophoretic method.For example, can before mass analysis, use liquid chromatography/mass spectrometry method (LC/MS) to implement such sample analysis.In addition, can use any chromatography separating method that is suitable for separating the goal analysis thing.The suitable chromatogram and the non-limiting example of electrophoresis separating method were described in this article.

In some embodiments, described method can be implemented with digestion and separating step.Though these steps are chosen wantonly, often they are implemented together, for example, thereby proteinic going up is in harmonious proportion when reducing in carrying out Proteomic analysis mensuration cell.In some embodiments, each step (no matter being with or without digestion and/or separating step) of method can be repeated one or many, thus one or more analytes in every kind of sample of one or more other analytes in evaluation and/or the quantitative sample or two or more samples (sample that comprises the labelled reagent institute mark that utilizes the upholder joint).Whether there is the calibration standard thing per sample in the mixture, quantitatively can being labeled analyte and carrying out of specific analyte with respect to other, perhaps specific analyte quantitatively can be absolute.

The analyte relevant with selected ion measured in the analysis of quality (rough quality or absolute mass) that as previously mentioned, can be by the antithetical phrase fragment ions.A kind of such method is described in the part of title for " analyte determination that is undertaken by the computer-Aided database analysis ".In case analyte is measured, then be can be the out of Memory of determining relevant sample mixture the basis is provided about the information of the information of every kind of unique report rough quality of thing ionic in second mass analysis and relative quantity and sub-fragment ions quality.

Can measure report thing ionic relative quantity by the peak intensity in the mass spectrum.In some embodiments, can measure every kind of unique report thing ionic amount by peak height or the peak width (or peak area) of utilizing spectrometer analysis gained report thing ion (characteristic ion).Because can utilize different labelled reagents that every kind of sample is carried out mark, and every kind of labelled reagent all can comprise and produces the unique report of unique report thing ionic thing part (described unique report thing ion can be associated with the specific otherness mark sample that is used to prepare sample mixture), therefore measures the sample (the report thing ion of selected analyte is by it) that different report thing ions can be used for identifying the otherness mark in second mass analysis.Under visible a plurality of report thing ionic situations (for example according to multiple method of the present invention), every kind of unique report thing ionic relative quantity can be reported the thing ion and measures with respect to other.Because every kind of unique report thing ionic relative quantity being measured in second mass analysis can be associated with the relative quantity of analyte in the sample mixture, and because be used to prepare the ratio of the sample of sample mixture is known, so can measure the relative quantity (being often expressed as concentration and/or amount) of analyte in the sample (its combination and form sample mixture) of every species diversity mark.In addition, be that (for example described analyte is the product of DeR under the situation of by product of another kind of purpose compound at the analyte of being measured, such as being under the situation of the peptide that forms by protein digestion at analyte), the quantitative information of analyte can be associated with composition in the original differences mark sample.

As mentioned above, can repeat this analysis one or many, thereby obtain the relative quantity of one or more other the tested analyte in every kind of sample (its combination and form sample mixture) at selected ion with different mass-to-charge ratioes.In addition, if desired, can carry out the correction of the isotopic abundance of natural or artificial generation to the peak intensity relevant with every kind of specific characteristic ion, be described in the part of " analyte relatively and absolute quantitation " at title as the front.

In some embodiments, described analyte can be the peptide in sample or the sample mixture.But the analysis of peptide in sample or the sample mixture be can be used to the amount (being often expressed as concentration and/or amount) of identification of protein in working sample or the sample mixture, and wherein the protein in one or more samples can be degraded before first mass analysis.In addition,, the information from different samples can be compared, such as being the relatively effect of proteinic amount in the cell that the matrix (it can influence cell growth, growth, differentiation and/or dead) of different concns is hatched for measuring purpose.The example of other indefiniteness can comprise the comparison to protein component expressed in ill or health tissues or the cell culture.This can be included in and utilize infective agent (such as bacterium or virus or other morbid state such as cancer) to infect the comparison of protein level expressed in back pair cell, tissue or the biofluid.In other examples, can carry out protein concn and change the effect of the research of (time-process) in time with protein component expressed in check pharmacological agent pair cell or the tissue.In other examples, from the information of the different samples of getting in time can be used for detecting or monitoring as the result's of disease (for example cancer) or infection the concentration of specified protein in tissue, organ or biofluid.These experiments can comprise one or more control samples.In some embodiments, described experiment can be used for measuring two or more above-mentioned interested character.

Under the calibration standard thing that in sample mixture, adds known quantity (being often expressed as concentration and/or amount) the situation of (comprising the uniqueness report thing part that links to each other with analyte), can utilize the described specific characteristic ionic amount relevant to measure the absolute magnitude (being often expressed as concentration and/or amount) of the middle analyte of every kind of sample (its combination and form sample mixture) with the calibration standard thing with selected mass-to-charge ratio.This is possible, because the amount of the analyte relevant with the uniqueness report thing ion of proofreading and correct standard substance in the sample mixture is known, and be labeled for the analyte for relevant with selected ion, the intensity of the characteristic ion of the described calibration standard thing of reference can be measured all specific characteristic ionic relative quantities.Because for every kind of unique report thing part (the report thing part that comprises described calibration standard thing), every kind of specific characteristic ionic relative quantity being surveyed is proportional with the amount of the analyte relevant with the sample of every species diversity mark (its combination and form sample mixture), therefore, reference can be determined the absolute magnitude (being often expressed as concentration and/or amount) of analyte in every kind of sample based on every kind of different characteristics ionic amount of the unique qualities of certain proportion (calculating with reference to the prescription that is used for the preparing product mixture).When needing, can carry out the correction of the isotopic abundance of natural or artificial generation to the peak intensity relevant with the characteristic ion of every kind of uniqueness.Such analytical procedure especially can be used for having the Proteomic analysis of the multiple sample of complicated character, particularly under the pre-separation that is labeled analyte before first mass analysis situation of (for example liquid chromatography is separated or electrophoretic separation).

For example, if sample mixture comprises the calibration standard thing of 100fmol/mL, and the specific characteristic ionic relative intensity relevant with described calibration standard thing is 1, and first other specific characteristic ionic relative intensity relevant with first sample be half and with second sample second relevant other specific characteristic ionic relative intensity is 2, the amount of analyte in the sample of the then described first otherness mark (its mixing and form sample mixture) (sample 1 of supposition equivalent and sample 2 mix and form described sample mixture) is that (0.5 * 100fmol/mL), the amount of analyte in the sample of the described second otherness mark (its mixing and form sample mixture) (sample 1 of supposition equivalent and sample 2 mix and form described sample mixture) is 200fmol/mL (2 * 100fmol/mL) to 50fmol/mL.And, for example, if analyte is the peptide with the specific protein qualitative correlation, can infer in sample 1 this proteinic amount be 50fmol/mL and in sample 2 this proteinic amount be 200fmol/mL.Therefore, the existence of calibration standard thing makes and can be in the sample of every species diversity mark (its mixing and form sample mixture) to carry out absolute quantitation to being labeled analyte (and its precursor) in some cases.

As previously mentioned, in some embodiments, but reference standard curve determination and every kind of corresponding absolute magnitude of unique report thing part with every kind of characteristic ion of unique qualities.Therefore, can be with reference to every kind of different characteristics ionic absolute magnitude and the absolute magnitude of institute's cls analysis thing of every kind of different samples in the working sample mixture with unique qualities.

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 one or more other analytes in every kind of sample (its combination and form sample mixture).In addition, if desired, can be as previously mentioned to carrying out the correction of the isotopic abundance of natural or artificial generation with every kind of unique relevant peak intensity of thing ion of reporting.

In some embodiments, can utilize by the labelled reagent of formula II ' (comprising its salt form and/or hydrate forms) representative and implement method as herein described:

Wherein W, M, J, K, R ₁, R ₂And X ₁As previously mentioned.

In some embodiments, can utilize at least a can be by formula III ' labelled reagent of (comprising its salt form and/or hydrate forms) representative implements methods described herein:

Wherein s, R ₁, R ₂And R ₁₁As previously mentioned.For example, can utilize and at least aly can implement described method to the labelled reagent of XII ' (comprising its salt form and/or hydrate forms) representative by formula V ' as follows:

R wherein ₁And R ₂As previously mentioned, symbol ^*Indicate and comprise when suitable ¹³C substitutes ¹²C, ¹⁵N substitutes ¹⁴N or ¹⁸O substitutes ¹⁶The isotopic enrichment site of O.

In some embodiments, can utilize and at least aly can implement described method by the labelled reagent of formula IV ' (comprising its salt form and/or hydrate forms) representative:

R wherein ₁₁As previously mentioned.For example, can utilize and at least aly can implement described method to the labelled reagent of XXIII ' (comprising its salt form and/or hydrate forms) representative by formula XV ' as follows:

Wherein, symbol ^*Indicate and comprise when suitable ¹³C substitutes ¹²C, ¹⁵N substitutes ¹⁴N or ¹⁸O substitutes ¹⁶The isotopic enrichment site of O.

In some embodiments, can utilize at least a can be by formula III ^*' labelled reagent of (comprising its salt form and/or hydrate forms) representative implements described method:

Wherein, R ₁₁As previously mentioned.For example, can utilize and at least aly can implement described method to the labelled reagent of MV ' (comprising its salt form and/or hydrate forms) representative by formula M ' as follows:

Wherein, symbol ^*Indicate and comprise when suitable ¹³C substitutes ¹²C, ¹⁵N substitutes ¹⁴N or ¹⁸O substitutes ¹⁶The isotopic enrichment site of O.

As previously mentioned, in some embodiments, can utilize different in nature set of tags of poor quality to implement described method.Therefore, in some embodiments, described method also can comprise at sample mixture or its fraction carries out first mass spectroscopy, and measures the relative intensity at the peak relevant with being labeled analyte.Because the evaluation to analyte can be determined by analyzing the daughter ion fragment, thereby therefore described method can comprise that also the ion that utilizes the dissociation energy fracture to be labeled analyte forms the daughter ion fragment and also identifies from the segmental analyte of described daughter ion.

In some embodiments, can utilize and at least aly can implement this method to the labelled reagent of XXXII ' (comprising its salt form and/or hydrate forms) representative by formula XXV ' as follows:

Wherein, symbol ^*Indicate and comprise when suitable ¹³C substitutes ¹²C, ¹⁵N substitutes ¹⁴N or ¹⁸O substitutes ¹⁶The isotopic enrichment site of O.

In some embodiments, can utilize and at least aly can implement this method to the labelled reagent of XXXXI ' (comprising its salt form and/or hydrate forms) representative by formula XXXV ' as follows:

Wherein, symbol ^*Indicate and comprise when suitable ¹³C substitutes ¹²C, ¹⁵N substitutes ¹⁴N or ¹⁸O substitutes ¹⁶The isotopic enrichment site of O.

In some embodiments, can utilize and at least aly can implement this method to the labelled reagent of MXIII ' (comprising its salt form and/or hydrate forms) representative by formula MVI ' as follows:

Wherein, symbol ^*Indicate and comprise when suitable ¹³C substitutes ¹²C, ¹⁵N substitutes ¹⁴N or ¹⁸O substitutes ¹⁶The isotopic enrichment site of O.

The protein group workflow

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

Utilize the example of Proteomic analysis, several at least spendable possibility workflows are arranged as indefiniteness.For ease of understanding following discussion, sometimes precursor protein and analyte peptides are distinguished.Yet, should be appreciated that in a plurality of embodiments, no matter be any or the two in protein and/or the peptide, all can think described analyte herein.

In one type workflow, described precursor protein can be digested the peptide analysis thing that can be labeled subsequently.In the workflow of another kind of type, described precursor protein can utilize labelled reagent to carry out mark, is digested the peptide analysis thing that is labeled then.In the workflow of another type, described precursor protein matter can be captured on the solid support, digestion, and what described upholder can be engaged then is peptide-labeled.Randomly, but the described peptide (flow throughpeptide) that flows through of mark also.In the workflow of another kind of type, precursor protein matter can capture on the solid support, be labeled, and can digest the peptide that is labeled with generation with upholder bonded protein then.Randomly can also analyze the described peptide that flows through.No matter workflow how, before analyzing, MS and MS/MS all carry out other sample preparation (for example analytical procedure) to being labeled peptide suitably the time.

Relate to and digest the exemplary operation flow process of mark subsequently

As an example, can there be " contrast " sample to be analyzed and " test " sample.If for example purpose is the peptide of analyzing in " contrast " and " test " sample protein (as analyte), so in some embodiments, can randomly reduce the protein (randomly seal halfcystine and use enzymic digestion) in the sample thus produce and can be labeled the analyte protein that is used for subsequent analysis.In some embodiments, analyte peptides mark (label) can not carried out further sample preparation.Mark anyway all can utilize the different labelled reagent report thing part (for example isometry and/or with the labelled reagent in the amount dystopy marker group) of unique qualities (each is self-contained have) to come the analyte in every kind of different samples of mark.

In some embodiments, may need further sample preparation before mark and/or after the mark.For example, can carry out separating step removing the uninterested peptide of some type, thereby reduce the complicacy of sample.Can will be labeled sample mix and obtain sample mixture.In some embodiments, can before mass spectroscopy, the described analyte peptides that is labeled be separated (for example high performance liquid chromatography (HPLC)).

Another exemplary embodiment comprises randomly seals and the step of the thiol group (it can relate to peptide and catch) of the halfcystine of regenerating.For avoiding doubt, self-evident, can handle other sample, prerequisite is can obtain other otherness marker with every kind of different sample or the sample fraction of encoding.

In some embodiments, can utilize enzymic digestion " contrast " sample and " test " sample, can capture on the solid support by the composition of cleavable linker then sample.For example, described upholder can comprise cleavable linker and the reaction active groups that can react with the structure division of peptide.

In some embodiments, can collect (rather than discarding) and flow through the peptide of upholder (because its not with the functional group reactions of upholder), utilize with the labelled reagent in amount dystopy and/or the different in nature labelled reagent group of poor quality its mark, and separate analysis or be labeled peptide analysis with what collect from upholder.Can utilize labelled reagent identical or different in the labelled reagent group that the peptide that flows through solid support is carried out mark.No matter labelled reagent how, all can randomly it be mixed with the sample mixture of analyzing by MS/MS.Also can analyze independently it.For the peptide that keeps on the upholder, can be when described peptide is still on upholder it be carried out mark or it is carried out mark when upholder cuts away at it.

Can utilize solid support to catch precursor protein.For example, can there be two kinds of samples that utilize parallel mode to handle.Can remove by cleaning and not contain halfcystine peptide partly also randomly with its collection (promptly flowing through).They also can be randomly analyzed by digestion, mark and/or with sample mixture, are perhaps analyzed separately.

The protein that described upholder engages can be digested.The peptide that contains halfcystine that can utilize labelled reagent that described upholder is engaged then carries out mark and cuts away from upholder.Also the peptide that contains halfcystine that described upholder can be engaged cuts down and then utilizes labelled reagent to carry out mark from upholder earlier.From different samples be labeled peptide (randomly comprising the peptide that is labeled that does not contain halfcystine part) can be mixed, handle and/or analyze with sample mixture, perhaps analyzed separately.

Can collect any peptide of implementing the result of digestion from the conduct of upholder release.Usually these peptides are the peptides that do not contain thiol group.Can utilize labelled reagent with these peptides mark and randomly mix, handle and/or analyze randomly, perhaps be analyzed separately with sample mixture.

Relate to the exemplary operation flow process that mark digests subsequently

No matter whether use upholder to catch analyte is used for analyzing, utilize the step of labelled reagent labelled analyte all can carry out before or after digestion or other chemical treatment, prerequisite is that described processing is not so that the quantitative invalid mode that is labeled analyte described herein changes marker.For protein example, the halfcystine of also reducible and sealing same protein utilizes the N-ε-lysine side-chain amino of labelled reagent mark same protein, then described protein digestion is become to be labeled peptide.

No matter its source is how, all can analyte be analyzed or can further handle (comprising the preparation sample mixture) to it to being labeled, for example by separating and/or handling by being fixed on the upholder.Being labeled protein can cut down from upholder, is digested then or be labeled protein still and between the upholder joint conditioning time to be digested.Under latter event, the digestion that upholder engages will discharge the peptide that does not contain the halfcystine part from upholder.These peptides can be collected and separate analysis randomly, perhaps analyze as a part that comprises the sample mixture that is labeled peptide (containing the halfcystine part) that the latter discharges.

During the described precursor protein of mark, the digestion pattern can make a change before being digested to peptide.For example, utilize tryptic digestion can expect main generation C-terminal arginine peptide, because N-is ε-lysine side-chain amino is labeled thing and modifies.Therefore, tryptic activity can be very similar to the activity of Arg-C.Therefore because only those C-terminal arginine peptides that also contain lysine side-chain can be labeled and can detect in mass spectrograph, so this provides the method for the complicacy that a kind of further reduction remains the sample further handling and/or analyze.

In some embodiments, reducible protein also utilizes labelled reagent (for example specific labelled reagent of mercaptan) labeling moiety Gelucystine, and what then described protein digestion is become to be used to analyze is labeled peptide.The described peptide analysis thing that is labeled can be analyzed or can be further processed, and for example, handles by separating and/or being fixed on the upholder.The reaction of the N-alpha-amino group that for example, can be by lysine side-chain and/or the functional group of N-epsilon-amino and upholder and will be labeled peptide and be fixed on the upholder.Upholder with the cleavable linker that is used for fixing the compound that contains amido functional group comprises that the upholder that contains the trityl connector is (referring to trityl chloride upholder (Trityl-Cl) or 2-chlorine trityl chloride upholder, can be available from Novabiochem (San Diego, CA)).This workflow is with aforementioned those are different.Can cut down from upholder being labeled analyte, further handle and/or analyze.Present method may not can bring substantial complicacy to reduce, because all estimated to contain at least one N-alpha-amino group by digestion peptide.

Previous examples is not to be intended to the various possible workflows of limit.They only are intended to exemplary illustration.About before digestion, carrying out the embodiment of mark, also be used in the further sample preparation before digesting.

Brief summary

Though being the mode emphasis by specific examples, aforementioned discussion carries out at Proteomic analysis with as the peptide and/or the protein measuring of analyte, but described principle is intended to contain the analyte of a lot of types, and aforementioned workflow does not need to carry out too much experiment applicable to the analyte of described a lot of types.Therefore, the scope of content disclosed herein is not to be intended to be limited on the described any of these specific examples.

IV. mixture

In some embodiments, the present invention relates to mixture (for example sample mixture).For example, described mixture can comprise the analyte that is labeled with the amount dystopy and/or the opposite sex of poor quality.The exemplary mixture and preparation method thereof and/or the analytical procedure that are labeled analyte are described in the part of " mark and analytical procedure " at above-mentioned title.

Can form mixture by all or part of mixing with two or more labeled reactant products, wherein utilize the different labelled reagents in the labelled reagent group that every kind of sample is carried out mark, wherein every kind of labelled reagent comprises the report thing part of (roughly) quality that has uniqueness.Can by two or more be labeled in the analyte every kind identify the uniqueness report thing part of described every kind of different labelled reagents from the labeled reactant in (i.e. source).Described labelled reagent can be the isotope-coded same amount dystopy (and/or isometry) and/or the labelled reagent of the opposite sex of poor quality.Therefore, to be labeled analyte can be with amount dystopy (and/or isometry) and/or the opposite sex of poor quality to two or more in the mixture.The labelled reagent relevant with these methods discussed in front with the character that is labeled analyte.

Analyte in the mixture can be any analyte.For example, the analyte in the mixture can be a peptide.Analyte in the mixture can be a protein.Analyte in the mixture can be peptide and protein.Analyte in the mixture can be a nucleic acid molecule.Analyte in the mixture can be a carbohydrate.Analyte in the mixture can be a lipid.Analyte in the mixture can be a prostanoid.Analyte in the mixture can be a lipid acid.Analyte in the mixture can be the carnitine class.Analyte in the mixture can be an amino acid.Analyte in the mixture can be a VITAMIN.Analyte in the mixture can be a steroid.Analyte in the mixture can be that quality is less than 1500 daltonian small molecules.Analyte in the mixture can comprise two or more different types of analytes (for example 1) lipid and steroid; Or 2) peptide, lipid, steroid and carbohydrate).

Mixture can comprise the analyte of any type of otherness mark, and described analyte contains novel report thing/connector part disclosed herein.For example, described mixture can comprise at least two kinds can be by formula I " analyte of the otherness mark of (comprising its salt form and/or hydrate forms) representative:

Wherein atom or group Y, J, K, R ₁, R ₂And X ₁Described in front and its characteristic disclosed, and wherein by the group of formula I^ representative

For all relevant with specific sample are labeled analyte all is identical, is different from any other and is derived from the isotopic enrichment site that is labeled analyte of different samples (its combination and form mixture) but its Chinese style I^ group comprises at least one.Z " be covalently bound analyte.

In some embodiments, two kinds be labeled in the analyte any all can be from different samples.In some embodiments, the formula I of every kind of different labelled analytes " group Y-J (described group can form report thing part) can be encoded uniquely in one or more isotopic enrichments site; make when the group J of group Y-J ruptures in mass spectrograph with key between the rest part that is labeled analyte; can form report thing ion; it is different from any other that derives from different samples (its combination and form mixture) and is labeled any report thing ion that analyte is correlated with that wherein said uniqueness reports that the thing ion can identify the described sample that analyte is originated that is labeled with unique qualities.Group Z " be covalently bound analyte.For every kind of different marker, some in the mixture are labeled analyte can be identical, and some are labeled analyte can be different.

In some embodiments, formula I^ group

Comprise and be labeled the same rough quality of analyte (it is relevant with specific sample), but this rough quality is different from any other that is derived from different samples (its combination and form mixture) and is labeled the rough quality of the relevant formula I^ group of analyte, and the rough quality of its Chinese style I^ group can be identified the described sample that analyte is originated that is labeled.

In some embodiments, described mixture can comprise at least two kinds can be by formula II " analyte of the otherness mark of (comprising its salt form and/or hydrate forms) representative:

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

In some embodiments, described mixture can comprise the analyte of at least two species diversity marks, wherein at least aly is labeled analyte by formula III " (comprising its salt form and/or hydrate forms) representative:

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

In some embodiments, described mixture can comprise the analyte of at least two species diversity marks, and the wherein at least a analyte that is labeled is by formula V ", VI ", VII ", VIII ", IX ", X ", XI " or XII " (comprising its salt form and/or hydrate forms) representative:

Wherein, symbol ^*Indicate and comprise when suitable ¹³C substitutes ¹²C, ¹⁵N substitutes ¹⁴N or ¹⁸O substitutes ¹⁶The isotopic enrichment site of O.

In some embodiments, described mixture can comprise the analyte of at least two species diversity marks, and the wherein at least a analyte that is labeled is by formula XXV ", XXVI ", XXVII ", XXVIII ", XXIX ", XXX ", XXXI " or XXXII " (comprising its salt form and/or hydrate forms) representative:

Wherein, symbol ^*Indicate and comprise when suitable ¹³C substitutes ¹²C, ¹⁵N substitutes ¹⁴N or ¹⁸O substitutes ¹⁶The isotopic enrichment site of O.

In some embodiments, described mixture can comprise the analyte of at least two species diversity marks, and the wherein at least a analyte that is labeled is by formula IV " (comprising its salt form and/or hydrate forms) representative:

R wherein ₁₁And Z " as mentioned above.

In some embodiments, described mixture can comprise the analyte of at least two species diversity marks, and the wherein at least a analyte that is labeled is by formula XV ", XVI ", XVII ", XVIII ", XIX ", XX ", XXI ", XXII " or XXIII " (comprising its salt form and/or hydrate forms) representative:

Wherein, symbol ^*Indicate and comprise when suitable ¹³C substitutes ¹²C, ¹⁵N substitutes ¹⁴N or ¹⁸O substitutes ¹⁶The isotopic enrichment site of O.

In some embodiments, described mixture can comprise the analyte of at least two species diversity marks, and the wherein at least a analyte that is labeled is by formula XXXV ", XXXVI ", XXXVII ", XXXVIII ", XXXIX ", XXXX " or XXXXI " (comprising its salt form and/or hydrate forms) representative:

Wherein, symbol ^*Indicate and comprise when suitable ¹³C substitutes ¹²C, ¹⁵N substitutes ¹⁴N or ¹⁸O substitutes ¹⁶The isotopic enrichment site of O.

In some embodiments, described mixture can comprise the analyte of at least two species diversity marks, wherein at least aly is labeled analyte by formula III ^*" (comprising its salt form and/or hydrate forms) representative:

R wherein ₁₁And Z " as mentioned above.

In some embodiments, described mixture can comprise the analyte of at least two species diversity marks, and the wherein at least a analyte that is labeled is by formula M ", MI ", MII ", MIII ", MIV " or MV " (comprising its salt form and/or hydrate forms) representative:

Wherein, symbol ^*Indicate and comprise when suitable ¹³C substitutes ¹²C, ¹⁵N substitutes ¹⁴N or ¹⁸O substitutes ¹⁶The isotopic enrichment site of O.

In some embodiments, described mixture can comprise the analyte of at least two species diversity marks, and the wherein at least a analyte that is labeled is by formula MVI ", MVII ", MVIII ", MIX ", MX ", MXI ", MXII " or MXIII " (comprising its salt form and/or hydrate forms) representative:

Wherein, symbol ^*Indicate and comprise when suitable ¹³C substitutes ¹²C, ¹⁵N substitutes ¹⁴N or ¹⁸O substitutes ¹⁶The isotopic enrichment site of O.

V. test kit

In some embodiments, the present invention relates to test kit.Described test kit can comprise described labelled reagent and one or more other reagent, container, enzyme, buffer reagent and/or operation instruction herein.Described test kit can comprise group and one or more other reagent, container, enzyme, buffer reagent and/or the operation instruction of two or more labelled reagents.For example, described test kit can comprise at least a selected other reagent to implement the method for one or more analytes in quantitative two or more different samples.For example, described test kit can comprise and is labeled the calibration standard thing, and the described calibration standard thing that is labeled contains the report thing part with unique rough quality.For example, described test kit can comprise and is labeled the calibration standard thing, and the described calibration standard thing that is labeled contains the marker part with unique rough quality.

Two or more labelled reagents in the test kit can be isometrys and/or isobaric.For example, one or more labelled reagents in the test kit can be the compounds (comprising the compound group) of as described earlier in this article formula 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 test kit can comprise as described earlier in this article have be labeled analyte (for example as the calibration standard thing): I with following formula ", 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 test kit.Described test kit can for example be used for the multiple analysis of one or more analytes of same sample or two or more different samples.

VI. exemplary indicia reagent

Should be appreciated that labelled reagent A shown below and B have represented a kind of in the many possible labelled reagent that can prepare and use separately.It is also understood that those skilled in the art only utilize normal experiment and content disclosed herein can easily make the labelled reagent with similar chemical structure.Therefore, it is illustrative that disclosed content is intended to, and is not to be intended to limit or to limit by any way.

The exemplary isotope-coded same amount dystopy group A that can prepare and the compound of B have been shown.Should be appreciated that labelled reagent group shown below represented a kind of in the many possible labelled reagent group that can prepare and use separately.It is also understood that those skilled in the art only utilize normal experiment and content disclosed herein can easily make the labelled reagent group with similar chemical structure.Therefore, it is illustrative that disclosed content is intended to, and is not to be intended to limit or to limit by any way.

Exemplary group A:

Exemplary group B:

The opposite sex group C exemplary isotope-coded of poor quality that can prepare and the compound of D have been shown.Should be appreciated that labelled reagent group shown below represented a kind of in the many possible labelled reagent group that can prepare and use separately.It is also understood that those skilled in the art only utilize normal experiment and content disclosed herein can easily make the labelled reagent group with similar chemical structure.Therefore, it is illustrative that disclosed content is intended to, and is not to be intended to limit or to limit by any way.

Exemplary group C:

Exemplary group D:

Other exemplary isotope-coded compound of the preparation of method shown in can utilizing is described in this specification sheets and accompanying drawing and claims.

Though described this instruction in conjunction with a plurality of embodiments, and do not meant that this instruction is limited to these embodiments.On the contrary, skilled person in the art will appreciate that this instruction contained various replacement schemes, change and equivalent.

Claims (73)

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

Wherein,

Y is 5,6 or 7 yuan of heterocycles, and it can be replacement or unsubstituted, and can randomly can link to each other with upholder with cutting, and wherein said heterocycle comprises at least one theheterocyclic nitrogen atom that links to each other with group J by covalent linkage;

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 ₂-(CK ' ₂) _m) _pThe group of-representative, wherein n is 0 or 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

Wherein,

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 ₂-(CR ' ₂) _m) _pThe group of-representative, it forms the ring of described two nitrogen-atoms of bridge joint, wherein q is 1～10 integer, each m is 1～5 integer independently of one another, p is 1～4 integer, each R ' 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

X ₁Be=O ,=S ,=NH or=NR ₇

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

Z is hydrogen or covalently bound analyte; Wherein,

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

Each R ₃', R ₄' and/or R ₅' be alkylidene group, alkenylene, alkynylene, arylidene or alkyl arylene independently of one another; With

Described compound comprises at least one isotopic enrichment site.

2. the compound of claim 1, wherein group Y-J comprises at least one isotopic enrichment site.

3. the compound of claim 1 is wherein by formula I ^#The group of representative:

Comprise at least one isotopic enrichment site;

Wherein,

K be by formula-(CK ' ₂) _n-or-((CK ' ₂) _m-X ₂-(CK ' ₂) _m) _pThe group of-representative, wherein n is 0 or 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

Wherein,

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 ₂-(CR ' ₂) _m) _pThe group of-representative, it forms the ring of described two nitrogen-atoms of bridge joint, wherein q is 1～10 integer, each m is 1～5 integer independently of one another, p is 1～4 integer, each R ' 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

X ₁Be=O ,=S ,=NH or=NR ₇With

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

Wherein,

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

Each R ₄' and/or R ₅' be alkylidene group, alkenylene, alkynylene, arylidene or alkyl arylene independently of one another.

4. the compound of claim 1, wherein the group by formula Y-J representative comprises at least one isotopic enrichment site, by formula I ^#The group of representative

Comprise at least one isotopic enrichment site;

Wherein,

Y is 5,6 or 7 yuan of heterocycles, and it can be replacement or unsubstituted, and can randomly can link to each other with upholder with cutting, and wherein said heterocycle comprises at least one theheterocyclic nitrogen atom that links to each other with group J by covalent linkage;

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 ₂-(CK ' ₂) _m) _pThe group of-representative, wherein n is 0 or 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

Wherein,

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 ₂-(CR ' ₂) _m) _pThe group of-representative, it forms the ring of described two nitrogen-atoms of bridge joint, wherein q is 1～10 integer, each m is 1～5 integer independently of one another, p is 1～4 integer, each R ' 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

X ₁Be=O ,=S ,=NH or=NR ₇With

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

Wherein,

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

Each R ₃', R ₄' and/or R ₅' be alkylidene group, alkenylene, alkynylene, arylidene or alkyl arylene independently of one another.

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

Wherein,

W is atom or the group that is used for replacing at least one M group on described 6 yuan of heterocycles, and be in described the above nitrogen of six-ring the neighbour, or contraposition, it is-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 ' 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 ₂-(CK ' ₂) _m) _pThe group of-representative, wherein n is 0 or 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

Wherein,

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 ₂-(CR ' ₂) _m) _pThe group of-representative, it forms the ring of described two nitrogen-atoms of bridge joint, wherein q is 1～10 integer, each m is 1～5 integer independently of one another, p is 1～4 integer, each R ' 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

X ₁Be=O ,=S ,=NH or=NR ₇

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

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

Each R " ' be H independently of one another ₂N-R ₉'-, H (R ₁₀) N-R ₉'-, (R ₁₀) ₂N-R ₉'-, HO-R ₉'-, HS-R ₉The cleavable linker that '-maybe can be connected this compound and upholder with cutting;

Z is hydrogen or covalently bound analyte;

Wherein,

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

Each R ₃', R ₄', R ₅', R ₆', R ₈' and/or R ₉' be alkylidene group, alkenylene, alkynylene, arylidene or alkyl arylene independently of one another.

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

Wherein,

S is 0～5 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 " ' and

Z is hydrogen or covalently bound analyte; 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 cleavable linker that described compound and upholder can be connected with cutting; And

Wherein,

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

Each R ₉' be alkylidene group, alkenylene, alkynylene, arylidene or alkyl arylene independently of one another.

7. the compound of claim 6, wherein said compound comprises its salt form and/or hydrate forms by formula V, VI, VII, VIII, IX, X, XI or XII representative:

Wherein,

^*Indicate and comprise when suitable ¹³C substitutes ¹²C, ¹⁵N substitutes ¹⁴N or ¹⁸O substitutes ¹⁶The isotopic enrichment site of O;

R ₁Be hydrogen or R ₆

R ₂Be hydrogen or R ₇With

Z is hydrogen or covalently bound analyte; Wherein,

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

8. the compound of claim 6, wherein said compound comprises its salt form and/or hydrate forms by formula XXV, XXVI, XXVII, XXVIII, XXIX, XXX, XXXI or XXXII representative:

Wherein,

^*Indicate and comprise when suitable ¹³C substitutes ¹²C or ¹⁵N substitutes ¹⁴The isotopic enrichment site of N;

R ₁Be hydrogen or R ₆

R ₂Be hydrogen or R ₇With

Z is hydrogen or covalently bound analyte; Wherein,

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

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

Wherein,

R ₁₁Be hydrogen, deuterium, methyl ,-C (H) ₂D ,-C (H) D ₂,-CD ₃Or-R " ' and Z be hydrogen or covalently bound analyte; 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 cleavable linker that described compound and upholder can be connected with cutting; And

Wherein,

Each R ₉' be alkylidene group, alkenylene, alkynylene, arylidene or alkyl arylene independently of one another; With

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

10. the compound of claim 9, wherein said compound comprises its salt form and/or hydrate forms by formula XV, XVI, XVII, XVIII, XIX, XX, XXI, XXII or XIII representative:

Wherein,

^*Indicate and comprise when suitable ¹³C substitutes ¹²C, ¹⁵N substitutes ¹⁴N or ¹⁸O substitutes ¹⁶The isotopic enrichment site of O; With

Z is hydrogen or covalently bound analyte.

11. the compound of claim 9, wherein said compound comprises its salt form and/or hydrate forms by formula XXXV, XXXVI, XXXVII, XXXVIII, XXXIX, XXXX or XXXXI representative:

Wherein,

^*Indicate and comprise when suitable ¹³C substitutes ¹²C or ¹⁵N substitutes ¹⁴The isotopic enrichment site of N; With

Z is hydrogen or covalently bound analyte.

12. the compound of claim 5, described compound is by formula III ^*Representative comprises its salt form and/or hydrate forms:

Wherein,

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

Z is hydrogen or covalently bound analyte;

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 cleavable linker that described compound and upholder can be connected with cutting; And

Wherein,

Each R ₉' be alkylidene group, alkenylene, alkynylene, arylidene or alkyl arylene independently of one another; With

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

13. the compound of claim 12, wherein said compound comprises its salt form and/or hydrate forms by formula M, MI, MII, MIII, MIV or MV representative:

Wherein,

^*Indicate and comprise when suitable ¹³C substitutes ¹²C, ¹⁵N substitutes ¹⁴N or ¹⁸O substitutes ¹⁶The isotopic enrichment site of O; With

Z is hydrogen or covalently bound analyte.

14. the compound of claim 12, wherein said compound comprises its salt form and/or hydrate forms by formula MVI, MVII, MVIII, MIX, MX, MXI, MXII or MXIII representative:

Wherein,

^*Indicate and comprise when suitable ¹³C substitutes ¹²C or ¹⁵N substitutes ¹⁴The isotopic enrichment site of N; With

Z is hydrogen or covalently bound analyte.

15. each compound in the claim 1 to 6,9 or 12, wherein said compound comprises two or more isotopic enrichment sites.

16. each compound in the claim 1～15, wherein Z is a hydrogen.

17. each compound in the claim 1～15, wherein Z is peptide and/or protein.

18. each compound in the claim 1～15, wherein Z is prostaglandin(PG), lipid acid, carnitine, carbohydrate, lipid, amino acid, VITAMIN or steroid.

19. each compound in the claim 1～15, wherein said compound are the calibration standard things.

20. a method, it comprises:

A) make different labelled reagents reactions in two or more samples and the labelled reagent group, wherein every kind of sample comprises one or more reactive behavior analytes, thereby form the sample of two or more otherness marks, described every species diversity mark sample comprises one or more and is labeled analyte, different labelled reagents in wherein said group comprise its salt form and/or hydrate forms by formula I ' representative:

Wherein,

Y is 5,6 or 7 yuan of heterocycles, and it can be replacement or unsubstituted, and can randomly can link to each other with upholder with cutting, and wherein said heterocycle comprises at least one theheterocyclic nitrogen atom that links to each other with group J by covalent linkage;

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 ₂-(CK ' ₂) _m) _pThe group of-representative, wherein n is 0 or 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

Wherein,

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 ₂-(CR ' ₂) _m) _pThe group of-representative, it forms the ring of described two nitrogen-atoms of bridge joint, wherein q is 1～10 integer, each m is 1～5 integer independently of one another, p is 1～4 integer, each R ' 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

X ₁Be=O ,=S ,=NH or=NR ₇

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

Every kind of labelled reagent comprises at least one isotopic enrichment site; With

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

Each R ₃', R ₄' and/or R ₅' be alkylidene group, alkenylene, alkynylene, arylidene or alkyl arylene independently of one another; And

B) mix the sample of two or more described otherness marks or its part and one or more calibration standard things randomly, thereby produce sample mixture.

21. the method for claim 20, labelled reagent in wherein said group all is isobaric, every kind of different labelled reagent in wherein said group all has identical rough quality, but the group Y-J that wherein forms the report thing part of every kind of different labelled reagents is encoded uniquely in one or more isotopic enrichments site, make when the key between the remainder of the group J of described group Y-J and described labelled reagent ruptures the report thing ion that generation has unique qualities in mass spectrograph.

22. the method for claim 20, the labelled reagent in wherein said group all has different quality.

23. the method for claim 22, the group Y-J of every kind of different labelled reagents in wherein said group is encoded uniquely in one or more isotopic enrichments site, make when the key between the remainder of the group J of described group Y-J and described labelled reagent ruptures the report thing ion that generation has unique qualities in mass spectrograph.

24. the method for claim 21 also comprises:

C) described sample mixture or its fraction are carried out first mass spectroscopy;

D) ion that is labeled analyte from the selected mass-to-charge ratio of described first mass spectroscopy is applied dissociation energy, thereby form at least some selected ionic report thing ion and sub-fragment ions; And

E) selected ion, report thing ion and/or sub-fragment ions or its fraction are carried out second mass analysis.

25. the method for claim 24 also comprises:

F) measure every kind of rough quality and/or absolute mass of reporting the rough quality of thing ionic and relative quantity and some or all sub-fragment ions in second mass analysis.

26. the method for claim 25 also comprises:

G) measure the be labeled analyte relevant by analyzing sub-fragment ions with selected mass-to-charge ratio.

27. the method for claim 26 also comprises:

H) under different selected mass-to-charge ratioes to the selected ion repeating step (d) to (f) of described otherness labelled analyte or (d) to (g) one or many.

28. the method for claim 27 also comprises:

I) repeating step (c) to (f), (c) to (g) or (c) to (h) one or many are used the different fractions of described sample mixture at every turn.

29. each method in the claim 26 to 28 also comprises:

Repeating step (c) to (I) one or many.

30. each method in the claim 20 to 29, wherein group Y replaces or unsubstituted morpholine, piperidines or piperazine part.

31. each method in claim 21 or 23, the every kind of report thing ion evaluation that wherein has unique qualities is labeled the sample that analyte is derived from for every kind.

32. each method in the claim 20 to 31, every kind of different labelled reagent in wherein said group and upholder in conjunction with and link to each other with described upholder by cleavable linker, make every kind of different samples and the upholder that is loaded with different labelled reagents in described group react; And wherein said method also is included in carries out step (b) and carries out before:

I) randomly clean upholder to remove not the sample composition with the reaction active groups reaction of labelled reagent;

Thereby ii) cut the sample that described cleavable linker discharges and collect two or more otherness marks, every kind of sample comprises one or more and is labeled analyte, and wherein relevant with the specific sample described analyte that is labeled is partly to identify and/or quantitative by the report thing with unique qualities that is attached thereto; And

Iii) randomly before will being labeled every kind of sample mix of analyte, collect the every kind of sample that is labeled analyte.

33. each method in the claim 20 to 32 also comprises:

C) with the composition of every kind of sample of at least a enzymic digestion with degrade partially or completely described sample or sample mixture.

34. each method in the claim 20 to 33 also comprises:

C) separate described sample mixture.

35. the method for claim 25, every kind of report thing ion wherein determining to have unique qualities in described second mass analysis is with respect to other report thing ionic relative quantity.

36. the method for claim 35, every kind of report thing ionic relative quantity with unique qualities that wherein will be relevant with the institute identification of analytes is associated with the amount that merges the every species diversity mark sample that forms described sample mixture, thereby determines the relative quantity at analyte described in every kind of sample of described two or more the otherness mark samples that merge the described sample mixture of formation.

37. the method for claim 35, wherein:

I) described sample mixture comprises the calibration standard thing that is used for institute's cls analysis thing of known quantity, wherein said calibration standard thing comprises the report thing part with unique qualities that is used for institute's cls analysis thing, and determines and every kind of corresponding every kind of report thing ionic absolute magnitude with unique qualities of unique report thing part according to the uniqueness report thing ionic amount relevant with described calibration standard thing; And

Ii) determine the absolute magnitude of institute's cls analysis thing in every kind of different samples of described sample mixture according to having every kind of unique qualities different report thing ionic absolute magnitude.

38. the method for claim 35, wherein:

I) determine and every kind of corresponding every kind of report thing ionic absolute magnitude of unique report thing part according to typical curve with unique qualities; And

Ii) determine the absolute magnitude of institute's cls analysis thing in every kind of different samples of described sample mixture according to having every kind of unique qualities different report thing ionic absolute magnitude.

39. the method for claim 21, wherein at least a labelled reagent is by formula III ' representative, comprise its salt form and/or hydrate forms:

Wherein,

S is 0～5 integer;

R ₁Be hydrogen, deuterium or R ₆

R ₂Be hydrogen, deuterium or R ₇With

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

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 cleavable linker that described compound and upholder can be connected with cutting;

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

Each R ₉' be alkylidene group, alkenylene, alkynylene, arylidene or alkyl arylene 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 formula V ', VI ', VII ', VIII ', IX ', X ', XI ' or XII ' representative:

Wherein,

^*Indicate and comprise when suitable ¹³C substitutes ¹²C, ¹⁵N substitutes ¹⁴N or ¹⁸O substitutes ¹⁶The isotopic enrichment site of O;

R ₁Be hydrogen or R ₆

R ₂Be hydrogen or R ₇With

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

41. the method for claim 21, wherein at least a labelled reagent comprises its salt form and/or hydrate forms by formula IV ' representative:

Wherein,

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

R " ' be H ₂N-R ₉'-, H (R ₁₀) N-R ₉'-, (R ₁₀) ₂N-R ₉'-, HO-R ₉'-, HS-R ₉The cleavable linker that '-maybe can be connected this compound and upholder with cutting; With

Wherein,

Each R ₉' be alkylidene group, alkenylene, alkynylene, arylidene or alkyl arylene independently of one another; With

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

42. the method for claim 41, wherein at least a labelled reagent comprises its salt form and/or hydrate forms by formula XV ', XVI ', XVII ', XVIII ', XIX ', XX ', XXI ', XXII ' or XXIII ' representative:

Wherein, ^*Indicate and comprise when suitable ¹³C substitutes ¹²C, ¹⁵N substitutes ¹⁴N or ¹⁸O substitutes ¹⁶The isotopic enrichment site of O.

43. the method for claim 21, wherein at least a labelled reagent is by formula III ^*' representative:

Wherein,

R ₁₁Be hydrogen, deuterium, methyl ,-C (H) ₂D ,-C (H) D ₂,-CD ₃Or-R " ', 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 cleavable linker that described compound and upholder can be connected with cutting;

Each R ₉' be alkylidene group, alkenylene, alkynylene, arylidene or alkyl arylene independently of one another; With

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

44. the method for claim 43, wherein at least a labelled reagent comprises its salt form and/or hydrate forms by formula M ', MI ', MII ', MIII ', MIV ' or MV ' representative:

Wherein, ^*Indicate and comprise when suitable ¹³C substitutes ¹²C, ¹⁵N substitutes ¹⁴N or ¹⁸O substitutes ¹⁶The isotopic enrichment site of O.

45. the method for claim 22 or 23 also comprises:

C) described sample mixture or its fraction are carried out first mass spectroscopy; With

D) measure the relative intensity at the peak relevant with being labeled analyte.

46. the method for claim 45 also comprises:

E) apply dissociation energy so that be labeled the ion fragmentation of analyte, thereby form the daughter ion fragment; With

F) identify described analyte by described daughter ion fragment.

47. the method for claim 45 or 46, wherein at least a labelled reagent comprises its salt form and/or hydrate forms by formula XXV ', XXVI ', XXVII ', XXVIII ', XXIX ', XXX ', XXXI ' or XXXII ' representative:

Wherein,

^*Indicate and comprise when suitable ¹³C substitutes ¹²C or ¹⁵N substitutes ¹⁴The isotopic enrichment site of N;

R ₁Be hydrogen or R ₆

R ₂Be hydrogen or R ₇With

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

48. the method for claim 45 or 46, wherein at least a labelled reagent comprises its salt form and/or hydrate forms by formula XXXV ', XXXVI ', XXXVII ', XXXVIII ', XXXIX ', XXXX ' or XXXXI ' representative:

Wherein,

^*Indicate and comprise when suitable ¹³C substitutes ¹²C or ¹⁵N substitutes ¹⁴The isotopic enrichment site of N.

49. the method for claim 45 or 46, wherein at least a labelled reagent comprises its salt form and/or hydrate forms by formula MVI ', MVII ', MVIII ', MIX ', MX ', MXI ', MXII ' or MXIII ' representative:

Wherein,

^*Indicate and comprise when suitable ¹³C substitutes ¹²C or ¹⁵N substitutes ¹⁴The isotopic enrichment site of N.

50. comprise at least two kinds of mixtures that are labeled analyte, wherein said at least two kinds of being labeled in the analyte derive from different samples, every kind in the wherein said mixture is labeled analyte by formula I " representative:

Comprise its salt form and/or hydrate forms; Wherein,

Y is 5,6 or 7 yuan of heterocycles, and it can be replacement or unsubstituted, and wherein said heterocycle comprises at least one theheterocyclic nitrogen atom that links to each other with group J by covalent linkage;

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 ₂-(CK ' ₂) _m) _pThe group of-representative, wherein n is 0 or 1～10 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

Wherein,

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 ₂-(CR ' ₂) _m) _pThe group of-representative, it forms the ring of described two nitrogen-atoms of bridge joint, wherein q is 1～10 integer, each m is 1～5 integer independently of one another, p is 1～4 integer, each R ' 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

X ₁Be=O ,=S ,=NH or=NR ₇

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

Z " be covalently bound analyte;

Wherein,

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

Each R ₃', R ₄' and/or R ₅' be alkylidene group, alkenylene, alkynylene, arylidene or alkyl arylene independently of one another; With

Wherein, by the group of formula I^ representative

For all relevant with specific sample are labeled analyte is identical, but the group of its Chinese style I^ comprises at least one and is different from any other and is labeled the isotopic enrichment site of analyte, and described other is labeled analyte and derives from and merge the different samples that form described mixture.

51. the mixture of claim 50, the group Y-J that wherein forms report thing part is encoded uniquely in one or more isotopic enrichments site, make as the group J of group Y-J and when being labeled key between the rest part of analyte and in mass spectrograph, rupturing, generation has the report thing ion of unique rough quality, this report thing ion is different from any other and is labeled the relevant any report thing ion of analyte, wherein said other is labeled analyte and derives from the different samples that merge the described mixture of formation, and wherein said unique report thing ion can be identified the described sample that analyte is derived from that is labeled.

52. the mixture of claim 50 is wherein by the group of formula I^ representative

Comprise identical rough quality for all relevant with specific sample are labeled analyte, but described rough quality is different from any other and is labeled the rough quality of the relevant formula I^ group of analyte, described other is labeled analyte and derives from the different samples that merge the described mixture of formation, and the rough quality of its Chinese style I^ group can be identified the described sample that analyte is derived from that is labeled.

53. each mixture in the claim 50～52, wherein said to be labeled in the analyte one or more be peptide and/or protein.

54. each mixture in the claim 50～52, wherein said to be labeled in the analyte one or more be prostaglandin(PG), lipid acid, carnitine, carbohydrate, lipid, amino acid, VITAMIN and/or steroid.

55. each mixture in the claim 50～52 wherein at least aly is labeled analyte by formula III " representative, comprise its salt form and/or hydrate forms:

Wherein,

S is 0～5 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 " ' and

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'-, HS-R ₉The cleavable linker that '-maybe can be connected this compound and upholder with cutting;

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

Each R ₉' be alkylidene group, alkenylene, alkynylene, arylidene or alkyl arylene independently of one another.

56. the mixture of claim 51, the wherein at least a analyte that is labeled is by formula V ", VI ", VII ", VIII ", IX ", X ", XI " or XII " representative, comprise its salt form and/or hydrate forms:

Wherein,

^*Indicate and comprise when suitable ¹³C substitutes ¹²C, ¹⁵N substitutes ¹⁴N or ¹⁸O substitutes ¹⁶The isotopic enrichment site of O;

R ₁Be hydrogen or R ₆

R ₂Be hydrogen or R ₇With

Z " be covalently bound analyte; Wherein

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

57. the mixture of claim 52, the wherein at least a analyte that is labeled is by formula XXV ", XXVI ", XXVII ", XXVIII ", XXIX ", XXX ", XXXI " or XXXII " representative, comprise its salt form and/or hydrate forms:

Wherein,

^*Indicate and comprise when suitable ¹³C substitutes ¹²C or ¹⁵N substitutes ¹⁴The isotopic enrichment site of N;

R ₁Be hydrogen or R ₆

R ₂Be hydrogen or R ₇With

Z " be covalently bound analyte; Wherein

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

58. each mixture in the claim 50～52, the wherein at least a analyte that is labeled is by formula IV " representative, comprise its salt form and/or hydrate forms:

Wherein,

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

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'-, HS-R ₉'-or cleavable linker that described compound and upholder can be connected with cutting;

Each R ₉' be alkylidene group, alkenylene, alkynylene, arylidene or alkyl arylene independently of one another; With

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

59. the mixture of claim 51, the wherein at least a analyte that is labeled is by formula XV ", XVI ", XVII ", XVIII ", XIX ", XX ", XXI ", XXII " or XXIII " representative, comprise its salt form and/or hydrate forms:

Wherein,

^*Indicate and comprise when suitable ¹³C substitutes ¹²C, ¹⁵N substitutes ¹⁴N or ¹⁸O substitutes ¹⁶The isotopic enrichment site of O;

Z " be covalently bound analyte.

60. the mixture of claim 52, the wherein at least a analyte that is labeled is by formula XXXV ", XXXVI ", XXXVII ", XXXVIII ", XXXIX ", XXXX " or XXXXI " representative, comprise its salt form and/or hydrate forms:

Wherein,

^*Indicate and comprise when suitable ¹³C substitutes ¹²C or ¹⁵N substitutes ¹⁴The isotopic enrichment site of N;

Z " be covalently bound analyte.

61. each mixture in the claim 50～52 wherein at least aly is labeled analyte by formula III ^*" representative, comprise its salt form and/or hydrate forms:

Wherein,

R ₁₁Be hydrogen, deuterium, methyl ,-C (H) ₂D ,-C (H) D ₂,-CD ₃Or-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'-, HS-R ₉The cleavable linker that '-maybe can be connected this compound and upholder with cutting;

Each R ₉' be alkylidene group, alkenylene, alkynylene, arylidene or alkyl arylene independently of one another; With

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

62. the mixture of claim 51, the wherein at least a analyte that is labeled is by formula M ", MI ", MII ", MIII ", MIV " or MV " representative, comprise its salt form and/or hydrate forms:

Wherein,

^*Indicate and comprise when suitable ¹³C substitutes ¹²C, ¹⁵N substitutes ¹⁴N or ¹⁸O substitutes ¹⁶The isotopic enrichment site of O;

Z " be covalently bound analyte.

63. the mixture of claim 52, the wherein at least a analyte that is labeled is by formula MVI ", MVII ", MVIII ", MIX ", MX ", MXI ", MXII " or MXIII " representative, comprise its salt form and/or hydrate forms:

Wherein,

^*Indicate and comprise when suitable ¹³C substitutes ¹²C or ¹⁵N substitutes ¹⁴The isotopic enrichment site of N;

Z " be covalently bound analyte.

64. a test kit, it comprises according to each at least a compound in the claim 1～19.

65. the test kit of claim 64, it also comprises at least a other reagent, and described other reagent is selected to the mensuration of carrying out one or more analytes in quantitative two or more different samples.

66. the test kit of claim 64, wherein said compound be comprise report thing part with unique rough quality be labeled the calibration standard thing.

67. the test kit of claim 64, wherein said compound be comprise marker part with unique rough quality be labeled the calibration standard thing.

68. the compound of formula A representative:

Wherein said compound comprises at least one isotopic enrichment site.

69. the compound of formula B representative:

Wherein said compound comprises at least one isotopic enrichment site.

70. with the group of amount dystopy compound, it comprises the compound by formula AI, AII, AIII and AIV representative:

Wherein ^*Indicate and comprise when suitable ¹³C substitutes ¹²C, ¹⁵N substitutes ¹⁴N or ¹⁸O substitutes ¹⁶The isotopic enrichment site of O.

71. with the group of amount dystopy compound, it comprises the compound by formula BI, BII, BIII and BIV representative:

Wherein ^*Indicate and comprise when suitable ¹³C substitutes ¹²C, ¹⁵N substitutes ¹⁴N or ¹⁸O substitutes ¹⁶The isotopic enrichment site of O.

72. have the group of the structural similitude compound of different rough quality, it comprises the compound by formula AI and AV representative:

Wherein ^*Indicate and comprise when suitable ¹³C substitutes ¹²C or ¹⁵N substitutes ¹⁴The isotopic enrichment site of N.

73. have the group of the structural similitude compound of different rough quality, it comprises the compound by formula BI and BV representative:

Wherein ^*Indicate and comprise when suitable ¹³C substitutes ¹²C or ¹⁵N substitutes ¹⁴The isotopic enrichment site of N.

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