CN109187940A - The preparation and application of a kind of isotopic tag reagent for Analysis of polysaccharides - Google Patents
The preparation and application of a kind of isotopic tag reagent for Analysis of polysaccharides Download PDFInfo
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Abstract
The invention discloses a kind of N- Analysis of polysaccharides method, with comprising MS/MS can the isotopic tag reagent containing quaternary amine of shear key and the reactive group that can be conjugated with N- polysaccharide mark N- polysaccharide.Also disclose a kind of quaternary amine containing isobaric tagging reagents, it is characterised in that: it includes following formulas: reporter group-balance group-reactive group, specific structure areWherein at least one of position 1-3 includes that isotope atom, reporter group and balance group are keyed by MS/MS shearing, and R is the reactive group that can be conjugated with polysaccharide.A kind of method for being synthetically prepared the isotopic tag reagent containing quaternary amine is also disclosed.
Description
Technical field
The present invention relates to biomolecule analysis reagent fields, are especially used for biomolecule quantitative analysis, particularly for
The preparation and application of a kind of isotopic tag reagent of Analysis of polysaccharides.
Background technique
Carbohydrate is one of the macromolecular to play an important role during biological life, and various glycoconjugates are them
One of most common modification of its macromolecular (Dove, Biotechnol.2001,19,913-917；Dennis, J.W.et al,
M.Cell 2009,139,1229-1241).For example, many pharmaceutical grade proteins (such as monoclonal antibody) have various polyose modifications,
The composition and stoichiometry of its polysaccharide can influence the stability and effect (Walsh, G.Jefferis, R., Nat. of its molecular structure
Biotechnol, 2006,24,1241-1252).Therefore, the qualitative and quantitative determination of polysaccharide is for biomedical and biotechnology
Using be all of great significance (Marino, K.Nat.Chem.Biol.2010,6,713-723).However, due to it chemically
The complexity of matter and structure, the research of polysaccharide and its comprehensive analysis lag far behind other biomolecule.
Polysaccharide (also known as: carbohydrate or glycan) is in bioprocess such as protein import, cell and cell-cell communication
With the important biomolecule of one kind to play a crucial role in immune response, and their exception with include cancer, it is dull-witted and from
Many diseases such as body immunological disease are related, what the effect and stability of therapeutic biological agent such as monoclonal antibody were also carried by them
The influence of polysaccharide.For these reasons, Analysis of polysaccharides (controlling for quality, medical diagnosis on disease etc.) is in academic research, pharmaceuticals industry
It is greatly worth with having in health care.However, polysaccharide is a lack of, UV absorbs and the hydrophily of easily ionizable is not divided in mass spectrum (MS)
Son.Their structure is that height is heterogeneous, because each polysaccharide can have multiple stereoisomers.In addition, polysaccharide biology closes
At the process for being non-template-driven so that their the Nomenclature Composition and Structure of Complexes it is difficult to predict.
In order to overcome these obstacles, Analysis of polysaccharides was usually derivatized special in various analysis platforms to improve them in the past
It is the performance (Ruhaak, L.R.et al.Anal.Bioanal.Chem.2010,397,3457-3481) in mass spectrum (MS).With
Fluorescence 2- aminobenzoic acid (2-AA) and 2- aminobenzamide (2-AB) label can make them compatible with fluorescence detection, and increase
Its strong signal (Bigge, J.C.et al.Anal.Biochem.1995,230,229-238) in MS.But since peak is total
Elution, fluorescent marker is extremely limited in the polysaccharide sample of quantitative complexity.The permethylated of hydroxyl can improve on polysaccharide
Their Mass Spectrometer Method, and the high quality MS2 spectrum for being used for structure elucidation is generated, but realize accurately quantitatively still to have and choose
War property and sample analysis reproducibility it is lower (Kang, P.et al.Rapid Commun.Mass Spectrom. 2005,19,
3421-3428.In recent years (Prien, J.M.et al.Anal.Chem.2010,82,1498-1508) is marked with 2-AA/2-AB in
It is obtained with permethylated (Alvarez-Manilla, G.et al.Glycobiology 2007,17,677-687) more and more
Concern to help polysaccharide to characterize and quantitative (Bowman, M.J.et al..Anal.Chem.2007,79,5777-5784).
There are two kinds of stable isotope labeling method, quality displacement and isobar label.Mass spectrum shift reagent
Label makes the analyte from different samples generate the difference in quality of several dalton on its precursor mass (MS1), and
Quantitative analysis is realized in intensity by comparing the corresponding mass spectra peak of MS1.On the contrary, isobar label has identical chemical structure
And molecular weight, but the position of various heavy nucleus is different in its various isomery molecule.Therefore, once sample is by one group of isobar mark
Label label, they do not show quality difference in MS1.In MS/MS fragmentation, a series of low-quality report ions of difference are generated
For quantitative.Compared with quality shifts label, presently commercially available isobar label, such as Tandem mass label (TMT) 10-plex
Allow simultaneous quantitative up to 10 samples (McAlister, G.C.et al.Anal.Chem.2012,84,7469-7478), and
And more complexity will not be introduced in 1 spectrum of MS, it is accumulated in together by the signal from multiple samples to increase inspection
Survey sensitivity.Therefore, extensive quantitative protein (Zhang, J. are had been widely used for；Wang,Y.；Li,S.Anal.
Chem.2010,82,7588-7595；Zeng,D.；Li,S.Chem.Commun.2009,3369-3371；Chen,Z. et
Al.Anal.Chem.2012,84.2908-2915) and small molecule metabolites (Yuan, W.et al.Proteome Res.2011,
10,5242-5250；Yuan,W.et al.Anal.Chem.2012,84,2892-2899).
Had experiment attempt for isobar label to be used for polysaccharide it is quantitative (Yang, S.et al.Anal.Chem.2013,
85, 8188-8195；Hahne,H.et al.Anal.Chem.2012,84,3716-3724).However, due to the sugar in polysaccharide
Glycosidic bond is easy to be broken in MS/MS fragmentation and compete with the generation of report ion, therefore is initially what polypeptide analysis designed
Isobar label is often unsuitable for Analysis of polysaccharides.As the method remedied, labeled polysaccharide must be with sodium chloride (NaCl)
It carries out being mixed for MS analysis, this, which is primarily due to Na+, can assist the fragmentation of isobar label to generate more reports
Accuse object ion.The problems caused by this method is the ionization that salt ion can seriously inhibit electric spray ion source (ESI), drop
The signal of polysaccharide in low ESI-MS.Polysaccharide can also form multiple H+/Na+ adducts, and molecule is complicated, and further drop
They low detection sensitivity.
Therefore, Analysis of polysaccharides field still needs new technology, especially for enhancing reagent and the side of polysaccharide quantitative analysis
Method.
Summary of the invention
Polysaccharide is used as and rises in multiple important biomolecule process such as protein imports, cell and cell-cell communication and immune response
The biomolecule of key effect, their structure and expression quantity and many complex diseases (such as cancer, dull-witted and autoimmunity
Disease etc.) it is closely related.Meanwhile most of protein drugs (such as monoclonal antibody) also carry polysaccharide, and it is intracorporal steady in people
Qualitative and curative effect is influenced by these polysaccharide.Therefore, Analysis of polysaccharides has a wide range of applications.For example, provided by the present invention
Method can be applied to following various scenes (but being not limited only to these scenes): (1) in the research and development and production process of protein drug,
The curative effect and quality control of protein drug are improved for analyzing polysaccharide；(2) to a major class congenital disorders of glycosylation
(congenital disorder of glycosylation) is diagnosed；(3) in exploitation polysaccharide vaccine (including AIDS etc.
A variety of viral diseases) during, for studying the validity of vaccine.
For these application, the invention discloses N- Analysis of polysaccharides method, the isotopic tag reagent containing quaternary amine and its
Preparation method.
The invention provides the following technical scheme:
The present invention relates to the application of the isotopic tag reagent containing quaternary amine for biomolecule analysis, and preparation and
Use the method for quaternary amine isotopic tag reagent.Quaternary amine isotopic tag reagent has unique application in polysaccharide molecule analysis,
It is quantitative especially by the polysaccharide of tandem mass spectrometry.
The present invention provides a kind of N- Analysis of polysaccharides method, shear key and can be conjugated with N- polysaccharide with comprising MS/MS
The isotopic tag reagent containing quaternary amine of reactive group mark N- polysaccharide.
As further technical solution of the invention, the isotopic tag reagent containing quaternary amine includes following formula: report
Announcement group-balance group-reaction group, wherein reporter group and balance group, which pass through MS/MS, can shear key connection.
As further technical solution of the invention, the isotopic tag reagent containing quaternary amine includes following formula knot
Structure: reporter group-balance group-reactive group, specific structure are
Wherein at least one of position 1-3 includes that isotope atom, reporter group and balance group can be cut by MS/MS
Key connection is cut, and R is the reactive group that can be conjugated with polysaccharide.
As further technical solution of the invention, the quality of the reporter group in the range of 176 to 179Da,
And the reagent contains 2 or 3 isotope atoms independently selected from 13C and 2H.
As further technical solution of the invention, the reactive group includes that can be sewed by reduction amination and polysaccharide
The reactive primary amine of conjunction.
The present invention also provides the methods that the sensitivity of enhancing polysaccharide is used for glycosyl analysis comprising with containing the same of quaternary amine
The plain tagging reagents in position mark polysaccharide.Wherein, the N- polysaccharide is prepared by the following:
(1) fixed on solid support to be obtained from people or non-human animal's seroglycoid comprising N- polysaccharide；
(2) chemical modification carries the N- polysaccharide of sialic acid, and chemical modification includes the carbodiimide coupling of sialic acid；
(3) N- polysaccharide is discharged from solid support.
Quantitatively realizing by two steps to quaternary amine label polysaccharide in the present invention: (1) fragments characteristic peak among MS/MSs is generated；
(2) polysaccharide is quantified according to the intensity of characteristic peak.To N- polysaccharide quantitative analysis, the quantitative analysis step includes segment
Change, quantitative two steps, the N- polysaccharide after label generates fragments characteristic peak in MS/MS, according to the intensity of characteristic peak to N- polysaccharide into
Row is quantitative.
As further technical solution of the invention, wherein Na+It is not used in the N- polysaccharide of quantitative analysis label.
The invention also discloses a kind of, and the isotopic tag reagent containing quaternary amine (is named as QABIT=Quanternary
Amine Based Isobaric Tag), the isotopic tag reagent containing quaternary amine includes following formula: reporter group-balance group-
Reactive group,
Specific structure is
Wherein at least one of position 1-3 includes that isotope atom, reporter group and balance group can be broken by MS/MS
Key connection is split, and R is the reactive group that can be conjugated with polysaccharide.
As further technical solution of the invention, R is the one of which of primary amine, hydrazides or aminooxy group.
As further technical solution of the invention, the isotopic tag reagent containing quaternary amine is the same amount containing quaternary amine
Dystopy tagging reagents.
As further technical solution of the invention, wherein range of the quality of the reporter group 176 to 179Da
It is interior, the reagent contain independently selected from13C and22 or 3 isotope atoms of H, comprising reduction amination and polysaccharide can be passed through
The reactive primary amine of conjugation.
As further technical solution of the invention, wherein the quaternary amine containing the different sequence label of same amount has structure
(I):
And wherein 3 methyl are13CHD2。
As further technical solution of the invention, wherein the quaternary amine containing isobaric label has structure
(I):
And wherein 1 carbon is13C, 3 methyl are CHD2。
As further technical solution of the invention, wherein the quaternary amine containing isobaric label has structure
(I):
And wherein 2 methyl are CHD2, 3 methyl are13CH3。
As further technical solution of the invention, wherein the quaternary amine containing the different sequence label of same amount has structure
(I):
And wherein 1 carbon is13C, 2 methyl are CHD2。
The invention also discloses a kind of isotopic tag Reagent Protocols being synthetically prepared containing quaternary amine, comprising the following steps:
(a) it reacts compound 1 with compound 2, obtains compound 3；
(b) make compound 3 and sodium carbonate and methyl Iod R, obtain compound 4；
(c) make compound 4 and β-thioacetic acid, potassium hydroxide solution reaction obtains compound 5；
(d) make compound 5 and acetic acidreaction, obtain compound 6；
(e) tertbutyloxycarbonyl (Boc) protecting group is removed using the ethyl acetate solvent of hydrogen chloride in compound 6, obtained
Compound 7；
(f) it reacts compound 7 with o- nitrobenzene sulfonyl chloride, obtains compound 8；
(g) make compound 8 and sodium carbonate and methyl Iod R, obtain compound 9；
(h) make compound 9 and β-thioacetic acid, potassium hydroxide solution reaction obtains compound 10；
(i) it reacts compound 10 with tertbutyloxycarbonyl -2- aminoacetaldehyde (reactant 11), obtains compound 12；
(j) make compound 12 and sodium carbonate and methyl Iod R, obtain compound 13；
(k) tertbutyloxycarbonyl (Boc) protecting group is removed using the ethyl acetate solvent of hydrogen chloride in compound 13, obtained
Final products compound 14；
Synthesis technology is as follows:
Wherein the IUPAC systematic name of compound 1-14 is as follows:
Compound 1:tert-butyl (4- (aminomethyl) benzyl) carbamate
Compound 2:2-nitrobenzenesulfonyl chloride
Compound 3:tert-butyl (4- (((2-nitrophenyl) sulfonamido) methyl) benzyl)
carbamate
Compound 4:tert-butyl (4- (((N-methyl-2-nitrophenyl) sulfonamido) methyl)
benzyl) carbamate
Compound 5:tert-butyl (4- ((methylamino) methyl) benzyl) carbamate
Compound 6:tert-butyl (4- ((N-methylacetamido) methyl) benzyl) carbamate
Compound 7:(4- ((N-methylacetamido) methyl) phenyl) methanaminium chloride
Compound 8:N-methyl-N- (4- (((2-nitrophenyl) sulfonamido) methyl) benzyl)
acetamide
Compound 9:N-methyl-N- (4- (((N-methyl-2-nitrophenyl) sulfonamido) methyl)
benzyl) acetamide
Compound 10:N-methyl-N- (4- ((methylamino) methyl) benzyl) acetamide
Compound 11:tert-butyl (2-oxoethyl) carbamate
Compound 12:tert-butyl (2- (methyl (4- ((N-methylacetamido) methyl) benzyl)
amino) ethyl)carbamate
Compound 13:2- ((tert-butoxycarbonyl) amino)-N, N-dimethyl-N- (4- ((N-
methylacet amido)methyl)benzyl)ethan-1-aminium
Compound 14:mono (N1, N1-dimethyl-N1- (4- ((N-methylacetamido) methyl) benzyl)
ethane-1,2-diaminium)monochloride
Wherein step (b), (d), (j) used in methyl iodide, propionic acid, methyl iodide is respectively selected from following reactant to (i)-(iv)
In one group: the reactant comprising isotope labelling:
(i)CH3I；CH3COOH；13CHD2I
(ii)CH3I；CH313COOH；CHD2I
(iii)CHD2I；CH3COOH；13CH3I
(iv)CHD2I；13CH3COOH；CH3I.
Specific synthesis technology are as follows: compound 2 is then added in mixed compound 1 and triethylamine in methylene chloride (DCM),
Reaction is stirred overnight under an argon atmosphere, after the reaction was completed, DCM is added, mixture is washed twice with HCl solution, be saturated
NaHCO3 solution washes twice, and primary with aqueous salt solu-tion, by organic layer anhydrous Na 2SO4 drying and passes through
Rotavap is removed, and obtains compound as white solid 3；Compound 3 is dissolved in DMF, and Na2CO3 solid is added.Then it is added
Methyl iodide.Reaction is stirred 2 hours in the dark.Check that reaction is completed by HPLC.After the reaction was completed, it is removed by Rotavap
Remove most of CH 3I and DMF.Residue is dissolved in EtOAc and saline solution, separates organic layer and with 60mL saline solution
Washing three times, is removed with the drying of Na 2SO 4 and by Rotavap, obtains yellowish solid chemical compound 4；Compound 4 is dissolved in
In KOH/CH3OH solution, β-thioacetic acid is added.Reaction is stirred overnight under an argon atmosphere.After the reaction was completed, it is reacting
There is white precipitate in mixture.After filtering precipitating, methanol is carefully removed by Rotavap.Water is added into residue, is used in combination
EtOAc is extracted three times, combined organic layer washed once with saturation NaHCO3 solution, saline solution=washed once will have
Machine layer is dry with Na2SO4 and is removed by Rotavap, obtains light yellow solid Compound 5；Compound 5 is dissolved in DCM,
Acetic acid is added, EDC is then added, reaction is stirred overnight under argon atmosphere, it is after the reaction was completed, first molten with saturated sodium bicarbonate
Liquid is washed twice, is then washed twice with hydrochloric acid solution.It after solvent seasoning, is removed with rotary evaporation, obtains compound as white solid 6；
Compound 6 is dissolved in 2M HCl/EtOAc, is stirred 2 hours, a large amount of white precipitates occurs, filters and is washed with ethyl acetate,
Compound as white solid 7 is obtained after drying；Mixed compound 7 and triethylamine in methylene chloride (DCM), are then added compound
2, reaction is stirred overnight under an argon atmosphere, after the reaction was completed, DCM is added, by mixture HCl (50mmol/L) solution
It washes twice, saturation NaHCO3 solution washes twice, and primary with aqueous salt solu-tion.Organic layer is done with anhydrous Na 2SO4
It is dry and by Rotavap remove, obtain faint yellow solid compound 8；Compound 8 is dissolved in DMF, and it is solid that Na2CO3 is added
Body.Then methyl iodide is added, reaction is stirred 2 hours in the dark, checks that reaction is completed by HPLC, after the reaction was completed, leads to
It crosses Rotavap and removes major part CH 3I and DMF, residue is dissolved in EtOAc and saline solution, separate organic layer and use salt
Aqueous solution washs three times, is removed with the drying of Na 2SO 4 and by Rotavap, obtains yellowish solid chemical compound 9；By compound
9 are dissolved in KOH/CH3OH solution, and β-thioacetic acid is added, and reaction is stirred overnight under an argon atmosphere, after the reaction was completed,
After having white precipitate, filtering to precipitate in reaction mixture, methanol is carefully removed by Rotavap.Water is added into residue,
And three times with EtOAc extraction, combined organic layer washed once with saturation NaHCO3 solution, aqueous salt solu-tion is primary, will
Organic layer is dry with Na2SO4 and is removed by Rotavap, obtains light yellow solid Compound 10；By compound 10 be dissolved in containing
G of compound 11 is added in the methanol solution of acetic acid, and NaCNBH3 is dissolved in methanol, is slowly added into, after reaction stirring 2 hours, with rotation
Evaporimeter removes methanol.Semi-saturation sodium bicarbonate solution is added, is extracted with ethyl acetate 3 times, the organic phase saturation after merging
After salt washing, organic solvent is removed, uses methylene chloride and ethyl acetate as irrigation, is purified on silica gel column chromatography, obtained
Compound as white solid 12；Compound 12 is dissolved in DMF, and Na2CO3 solid is added, methyl iodide is then added, reaction is existed
It is stirred 2 hours in dark, checks that reaction is completed by HPLC, after the reaction was completed, most of iodomethane is removed by Rotavap
And DMF, solid purifies on silica gel column chromatography more than obtains compound as white solid 13；Compound 13 is dissolved in 2M HCl/
EtOAc is stirred 2 hours, a large amount of white precipitates occurs, is filtered and is washed with ethyl acetate, obtains white solid chemical combination after dry
Object 14；The synthesis of the QABIT of isotope labelling is identical as the synthesis of unlabelled compound 14, but from synthesis compound 4, changes
Object 6 is closed, uses following four groups of isotope labelling reactants: (CH3I, CH3COOH, 13CHD2I) during compound 13；
(CH3I, CH313COOH, CHD2I)；(CHD2I, CH3COOH, 13CH3I)；(CHD2I, CH313COOH, CH3I).
The present invention embodies novel technical solution compared with the prior art, possessed advantage and good effect, it be by
The direct bring certainty effect of technical characteristic.The invention has the following beneficial effects: (1) since the polysaccharide that quaternary amine marks carries not
The permanent positive charge influenced by solution acid alkalinity, the signal in mass spectrum greatly enhance, to greatly improve more
The sensitivity of glycan analysis increases the chance that rare polysaccharide is detected；(2) multiple polysaccharide samples can be analyzed simultaneously, significantly
The flux of Analysis of polysaccharides is improved, while improving the reproducibility of Analysis of polysaccharides.It, can be simultaneously to 4 in example of the invention
A sample is analyzed.But by simply extending, the present invention can be analyzed up to 10 samples；(3) saliva is carried
The N- polysaccharide of liquid acid is unstable usually in mass spectral analysis, and sialic acid often loses, and then causes to miss to the analysis of polysaccharide
Difference.Stability of the sialic acid in mass spectrum can be improved in method provided by the invention, helps precisely to analyze polysaccharide.
In conclusion Analysis of polysaccharides provided by the invention is compared with the conventional method, there is high sensitivity, high-throughput spy
Point is widely used in basic research, medicament research and development, the scenes such as disease diagnosis and therapy.
Detailed description of the invention
Fig. 1, with QABIT label N-acetyl-glucosamine (the first residue in GlcNAc, N- polysaccharide reduction end) and its
Fracture in MS2；(arrow indicates the broken site in MS2)
Fig. 2, the process for carrying out quantitative analysis to polysaccharide using QABIT；
The synthetic route of Fig. 3, QABIT reagent；
Fig. 4-Figure 13, QABIT mark the representative MS/MS map of N- polysaccharide, in figure arrow point out report ion (176,
177,178,179)；
Figure 14-Figure 16, QABIT are compared with commercially available aminoxyTMT marks polysaccharide；
The quantitative precision of Figure 17-Figure 19, QABIT.
Specific embodiment
With reference to the accompanying drawings and detailed description, the present invention is furture elucidated, it should be understood that following specific embodiments are only
For illustrating the present invention rather than limiting the scope of the invention.
The present invention relates to for biomolecule analysis the quaternary amine containing isotopic tag reagent and make and use containing
The method of the quaternary amine of isotopic tag reagent.Quaternary amine containing isotopic tag reagent is particularly useful for quantitative polysaccharide map.
As herein and used in the attached claims, unless the context clearly indicates otherwise, otherwise singular
" one ", "one" and "the" include plural object.Similar number refers to identical element, all figures with structural detail
Key all having the same, i.e. black squares indicate GlcNAc；Dark grey circle represents mannose；Light grey circle indicates gala
Sugar, diamond shape indicate sialic acid.
Herein with respect to its feature, aspect and example it is various illustrate the present invention in certain embodiments can be by structure
Making is to include, and forms or substantially by these features, some or all of aspect and example composition are used as its element and component
It is aggregated to constitute various further embodiments of the invention.The present invention is correspondingly imagined with various arrangements and combination in this hair
These features in bright range, aspect and example or selected one or more features, aspect and example.
Reagent of the invention can be used for marking or mark sample for passing through quantitative assay.It is used herein " quantitative "
Or " quantitative analysis " refers to the analysis of sample composition.Quantitatively allow to identify the measurable quality of the sample by this analysis, such as
The relative quantity of the element of sample, but regardless of the source of element in sample.Quantitative approach can include but is not limited to high pressure liquid phase color
It composes (HPLC), UV detection and mass spectrum (MS).
Present invention relates in general to the reagents comprising the different sequence label of same amount for quantitative biomolecule.Specifically, this hair
It is bright to be related to comprising the reagent for the isobar label that quantitatively sugar group is learned and saccharic composition is analysed.It is according to the present invention different containing same amount
Position label quaternary amine can be easily broken as glycosidic bond so that label polysaccharide can produce it is enough for analysis
Reporter ion (Fig. 1).Particularly, such analysis can be carried out in the case where not using Na+ or other metal ions.
Therefore, the present invention provides the preparation of the polysaccharide reactivity quaternary amine containing isobaric tagging reagents and its quantitative containing same
The purposes in the quaternary amine of the polysaccharide of dystopy label label is measured, such as passes through tandem mass spectrum (MS/MS).
In order to improve the report daughter ion intensity for the polysaccharide that the different sequence of same amount marks, it is expected that exploitation can be held as glycosidic bond
The isobar label of easy fracture, so that the polysaccharide of label can produce strong report daughter ion and quantify for accurate polysaccharide.
It has now been found that when MS/MS fragmentation, even if quaternary amine can easily lose it on nitrogen when it is coupled on polysaccharide
One of four substituent groups.Based on this observation, a kind of novel isobaric label is synthesized, QABIT has been named as: being based on
The isobar label of quaternary amine.This isobaric label is suitable for simultaneously that (Fig. 1 is specifically retouched to multiple polysaccharide samples quantitative
The structure and application method of QABIT reagent are stated).As it is used herein, term QABIT may include according to the present invention
What isobaric label or specific structure based on quaternary amine.
The present invention provides the quaternary amines of the isotopic tag reagent containing the reactive group comprising that can be conjugated with polysaccharide.
In embodiments, the quaternary amine containing isotopic tag reagent includes can be by the primary amine reaction of reduction amination and polysaccharide conjugation
Property group.
Quaternary amine according to the present invention comprising isotopic tag reagent generally comprises reporter group-balance group reactivity base
Group, wherein reporter group and balance group, which pass through MS/MS, can shear key connection, be easily broken off in MS/MS.
In embodiments, it is 176 to 181 in series that the structure of isobar label of the invention, which includes molecular weight ranges,
Dalton, the report molecule of preferably 176-179Da, the balance substance of compensation report molecular mass difference and reactive group and polysaccharide
Conjugation.In a particular embodiment, the quaternary amine containing isotopic tag reagent includes that can be conjugated by reduction amination and polysaccharide
Primary amine reaction group.
In some embodiments, isotopic tag reagent of the invention is containing the reagent of quadruple quaternary amine, and it includes tools
There are one group of four molecule of identical chemical structure and molecular weight, there is different stable nucleus, such as 13C and 2H in different location
Molecule.In other embodiments, the different sequence tagging reagents of same amount can be 6 weights or bigger, have the same position of weight in other position
Plain atom.
The substituent group in each of reporter group and balance group is selected, so that belonging to the substitution in reporter group
The mass change of the selection of base is attributed to the offset group of mass change of the selection of the substituent group in counterbalance.Therefore, reagent
Different isotope forms will add the identical summation of balance group quality with reporter group quality.
In MS/MS fragmentation, strong report can produce with the polysaccharide that the quaternary amine containing isotopic tag reagent marks
Ion is for accurately quantitative.In addition, there is the polysaccharide of permanent positively charged quaternary amine they can be enhanced in MS for cooperation
Ionization.Therefore, the significant enhancing of the detection sensitivity of polysaccharide, this is when analyzing low abundance polysaccharide or with the sample of limited supply
It is advantageous.Quaternary amine according to the present invention comprising isotopic tag reagent is for the Analysis of polysaccharides based on quaternary amine and/or to quantify
The first isobar label.These reagents use the chemistry similar with 2-AA/2-AB, therefore can change for 2-AA/2-
The perfect scheme of AB label and sample preparation.See, for example, U.S. Patent number 5,747,347, entire contents by reference simultaneously
Enter herein.Because quaternary amine is permanent positively charged, the quaternary amine of the polysaccharide containing isobar label easy electricity in MS
From, and it is significant improve its detection limit.
Even if isobar label is widely used for quantifying for peptide and small molecule metabolites, previously based on the glycosyl of tertiary amine
Quantitative trial has obtained limited success.U.S. Patent Publication No. 2015/0241437, is incorporated herein by reference；
Hahne,H.“Carbonyl-reactive Tandem mass tags for the proteome-wide
Quantification of N-linked glycans, Anal.Chem.84,3716-3724 (2012) are for example, commercial reagent
The polysaccharide of aminoxyTMT label needs to form metal ion adduct to show sufficiently strong report ion, for being broken
Shi Dingliang, this can cause the ion in MS to inhibit and reduce detection sensitivity.This no wonder because aminoxyTMT and its
It is quantitative that his the different sequence label of the same amount based on tertiary amine is initially designed to peptide.Since the glycosidic bond in polysaccharide is easier to break than peptide bond
It splits, therefore the polysaccharide of aminxy TM T label separates preferably between polysaccharide units, therefore generates the efficiency of report daughter ion
It is low.It was found that quaternary amine segment is simple as glycosidic bond in MS, to develop the quaternary amine containing isobar label,
This makes it possible to simultaneously from for the first time while realizing the global analysis of N- polysaccharide.
Isotopic tag containing quaternary amine of the invention has the structure of following general formula:
Wherein A-D is organic group.Quaternary amine containing isobaric label usually has a structure that
Report group-balance group-reaction group
Wherein, for the purpose of Analysis of polysaccharides, the reactive group can be conjugated with polysaccharide.It is according to the present invention containing
The quaternary amine of isobaric label can have various substituent groups on the nitrogen of quaternary amine, and condition is reporter group and balance
Group is broken by MS/MS and is keyed.
The isotope of quaternary amine according to the present invention containing isobar label can be incorporated to various positions, and can be
Any of heavy isotope.
As example, quaternary amine structure can be expressed as follows 4 and retry agent:
Wherein heavy isotope can be broken in any position in 1-4, reporter group and balance group by MS/MS
Key connection, and R is the reactive group that can be conjugated with polysaccharide.Reactive group, which can be, to be sewed by reduction amination and polysaccharide
The primary amine of conjunction.Reactive group can also be aminooxy group, hydrazides or other groups with aldehyde reaction.Isotope may include 15N,
13C and 2H.As an example, this structure can be any structure of following formula:
As shown in the table (isotope is shown with underscore):
| 1 | 2 | 3 | |
| 176 | C | CH3 | 13CHD2 |
| 177 | 13C | CH3 | CHD2 |
| 178 | C | CHD2 | 13CH3 |
| 179 | 13C | CHD2 | CH3 |
According to another aspect of the present invention, the isotopic tag reagent containing quaternary amine has following formula:
Wherein at least one of position 1-3 may include isotope atom, and reporter group and balance group pass through MS/MS
Fracture key connection, and R is reactive group and can be conjugated with polysaccharide.Reactive group can be can by reduction amination with
The primary amine of polysaccharide conjugation.Reactive group can also be aminooxy group, hydrazides or other groups with aldehyde reaction.
In one embodiment of the invention, the quaternary amine containing isobaric tagging reagents with following formula is provided
Report group-balance group-reaction group
Wherein the reagent has structure (I):
In such embodiments, reporter group and balance group are broken by MS/MS is keyed, and reporter group has
176 to 179Da molecular weight, reagent contain 2 or 3 isotope atoms for being independently selected from 13C and 2H, 2H, and reactive base
Group is comprising that can pass through the reactive primary amine of reduction amination and polysaccharide conjugation.
In the present invention, the quaternary amine containing isotopic tag reagent has substituent group.Therefore, in the reality according to structure (I)
It applies in scheme, 3 methyl are 13CHD2.In another embodiment, 3 methyl are CHD2, and 1 carbon is 13C,
In another embodiment, 2 methyl are CHD2, and 3 methyl are 13CH3, in another embodiment, 2
Methyl be 13CHD2.
Another aspect of the present invention provides a kind of N- Analysis of polysaccharides method comprising can be broken with containing comprising MS/MS
The label N- polysaccharide of the quaternary ammonium labelled reagent of key and the reactive group that can be conjugated with N- polysaccharide mark N- polysaccharide, polysaccharide.Root
According to the one aspect of this method, the quaternary amine containing isobaric tagging reagents includes following formula:
Report group-balance group-reaction group
Wherein reporter group and balance group, which pass through MS/MS, can shear key connection.
Wherein heavy isotope can be broken in any position in 1-3, reporter group and balance group by MS/MS
Key connection, and R is the reactive group that can be conjugated with polysaccharide.Reactive group, which can be, to be sewed by reduction amination and polysaccharide
The primary amine of conjunction.Reactive group can also be aminooxy group, hydrazides or other groups with aldehyde reaction.Isotope may include 13C and
2H。
In some embodiments, the quality of reporter group is in the range of 176 to 179Da, and reagent contains independence
Ground is selected from 2 or 3 isotope atoms of 13C and 2H.
In the other aspects of N- Analysis of polysaccharides method according to the present invention, reactive group includes that can pass through reduction amination
With the reactive primary amine of polysaccharide conjugation.
In the other aspects of N- Analysis of polysaccharides method according to the present invention, the method also includes the N- of quantitative analysis label
Polysaccharide (Fig. 2).
In the other aspects of N- Analysis of polysaccharides method according to the present invention, N- polysaccharide is obtained by following steps
(1) the fixed glycoprotein comprising N- polysaccharide on solid support；
(2) N- polysaccharide described in chemical modification；With
(3) N- polysaccharide is discharged from solid support.
According to these aspects, protein such as glycoprotein and solid support are conjugated, and unconjugated molecule can be by
It washes away.Glycocalix enzymatically modifying on immobilization glycoprotein passes through chemical reaction modification.Then, it is discharged from solid support more
Sugar is for analyzing.In these areas, solid support can be any material for becoming known for this purposes, such as polymeric beads
Or resin or controlled pore glass pearl.
In the other aspects of N- Analysis of polysaccharides method according to the present invention, the glycoprotein is obtained from biological sample, such as people
Or non-human animal's serum.As used herein, term " biological sample " or " biofluid " include but is not limited to from it is living or with
The substance of any amount of the subject of preceding work.Such substance includes but is not limited to blood, serum, blood plasma, urine, cell, organ,
Tissue, bone, marrow, lymph, lymph node, synovial tissue, CSF, cartilage cell, synovial macrophages, endothelial cell and skin.?
In preferred embodiment, fluid is blood or serum.
In the other aspects of N- Analysis of polysaccharides method according to the present invention, N- polysaccharide carrying sialic acid, and N- polysaccharide
Chemical modification includes the carbodiimide coupling of sialic acid.
In the other aspects of N- Analysis of polysaccharides method according to the present invention, quantitative analysis step includes fragmentation reagents and determines
Measure N- polysaccharide.
In the other aspects of N- Analysis of polysaccharides method according to the present invention, lytic reagent include MS/MS can shear key MS/
MS cracking.
In the other aspects of N- Analysis of polysaccharides method according to the present invention, Na+ is not used in the polysaccharide of quantitative analysis label.
Method of the invention including quantitative analysis step preferably includes to make reagent fracture and quantitative N- polysaccharide, more preferably makes
Reagent is broken with MS/ MS.Method of the invention for analyzing polysaccharide can be the case where not using Na+ or other metals
Lower progress.Such Na+ is not needed in quantitative analysis according to the method for the present invention, because quaternary amine segment is in MS as glucosides
Key is equally easy.
As shown in figure 3, the method for the quaternary amine invention additionally provides preparation comprising QABIT reagent, comprising the following steps:
(a) it reacts compound 1 with compound 2, obtains compound 3；
(b) make compound 3 and sodium carbonate and methyl Iod R, obtain compound 4；
(c) make compound 4 and β-thioacetic acid, potassium hydroxide solution reaction obtains compound 5；
(d) make compound 5 and acetic acidreaction, obtain compound 6；
(e) tertbutyloxycarbonyl (Boc) protecting group is removed using the ethyl acetate solvent of hydrogen chloride in compound 6, obtained
Compound 7；
(f) it reacts compound 7 with o- nitrobenzene sulfonyl chloride, obtains compound 8；
(g) make compound 8 and sodium carbonate and methyl Iod R, obtain compound 9；
(h) make compound 9 and β-thioacetic acid, potassium hydroxide solution reaction obtains compound 10；
(i) it reacts compound 10 with tertbutyloxycarbonyl -2- aminoacetaldehyde (reactant 11), obtains compound 12；
(j) make compound 12 and sodium carbonate and methyl Iod R, obtain compound 13；
(k) tertbutyloxycarbonyl (Boc) protecting group is removed using the ethyl acetate solvent of hydrogen chloride in compound 13, obtained
Final products compound 14.
Wherein the IUPAC systematic name of compound 1-14 is as follows:
Compound 1:tert-butyl (4- (aminomethyl) benzyl) carbamate
Compound 2:2-nitrobenzenesulfonyl chloride
Compound 3:tert-butyl (4- (((2-nitrophenyl) sulfonamido) methyl) benzyl)
carbamate
Compound 4:tert-butyl (4- (((N-methyl-2-nitrophenyl) sulfonamido) methyl)
benzyl) carbamate
Compound 5:tert-butyl (4- ((methylamino) methyl) benzyl) carbamate
Compound 6:tert-butyl (4- ((N-methylacetamido) methyl) benzyl) carbamate
Compound 7:(4- ((N-methylacetamido) methyl) phenyl) methanaminium chloride
Compound 8:N-methyl-N- (4- (((2-nitrophenyl) sulfonamido) methyl) benzyl)
acetamide
Compound 9:N-methyl-N- (4- (((N-methyl-2-nitrophenyl) sulfonamido) methyl)
benzyl) acetamide
Compound 10:N-methyl-N- (4- ((methylamino) methyl) benzyl) acetamide
Compound 11:tert-butyl (2-oxoethyl) carbamate
Compound 12:tert-butyl (2- (methyl (4- ((N-methylacetamido) methyl) benzyl)
amino) ethyl)carbamate
Compound 13:2- ((tert-butoxycarbonyl) amino)-N, N-dimethyl-N- (4- ((N-
methylacet amido)methyl)benzyl)ethan-1-aminium
Compound 14:mono (N1, N1-dimethyl-N1- (4- ((N-methylacetamido) methyl) benzyl)
ethane-1,2-diaminium)monochloride
Wherein step (b), (d), (j) used in methyl iodide, propionic acid, methyl iodide is respectively selected from following reactant to (i)-(iv)
In one group: the reactant comprising isotope labelling:
(i)CH3I；CH3COOH；13CHD2I
(ii)CH3I；CH313COOH；CHD2I
(iii)CHD2I；CH3COOH；13CH3I
(iv)CHD2I；13CH3COOH；CH3I.
Further illustrate that advantages and features of the invention, these embodiments are not necessarily to be construed as with reference to following specific embodiments
It limits the scope of the invention in any way, but in a particular application as the explanation of one embodiment of the invention.
Embodiment one: QABIT synthesizes
The synthetic route of QABIT as described in Figure 3；Using following specific synthetic route:
Mixed compound 1 (1.12g, 4.75mmol) and 1.5mL triethylamine in 30mL methylene chloride (DCM)
Then compound 2 (1g, 4.52mmol) is added in (10.8mmol).Reaction is stirred overnight under an argon atmosphere.Reaction is completed
Afterwards, 30mL DCM is added.Mixture is washed twice with 50mL HCl (50mmol/L) solution, 50mL saturation NaHCO3 solution is washed
It washs twice, and primary with 50mL aqueous salt solu-tion.It is removed organic layer anhydrous Na 2SO4 drying and by Rotavap, is obtained
To 1.7g compound as white solid 3 (4.03mmol, 89% yield).1H-NMR(CDCl3,400MHz)δ7.60-8.00(m,
4H), 7.10-7.20 (m, 4H), 4.28 (s, 2H), 4.22 (d, J=6.0Hz, 2H), 1.45 (s, 9H) .ESI-MS (M+H)+
=422.14, Cal. (M+H) +=422.14.
Compound 3 (1.7g, 4.40mmol) is dissolved in 10mL DMF, and it is solid that 2.8g Na2CO3 (26.4mmol) is added
Body.Then 1.37mL methyl iodide (22mmol) is added.Reaction is stirred 2 hours in the dark.Check that reaction is completed by HPLC.
After the reaction was completed, major part CH 3I and DMF are removed by Rotavap.Residue is dissolved in 50mL EtOAc and 50mL salt water
In solution.Separate organic layer and with 60mL aqueous salt solu-tion three times, it is dry with Na 2SO 4 and by Rotavap removing, obtain
To the yellowish solid chemical compound 4 (4.14mmol, 94% yield) of 1.8g.1H-NMR(CDCl3,400MHz)δ 7.60-8.00(m,
4H), 7.23-7.30 (m, 4H), 4.39 (s, 2H), 4.30 (d, J=6.0Hz, 2H), 2.76 (s, 3H), 1.45 (s, 9H)
.ESI-MS (M+H) +=436.13, Cal. (M+H) +=436.14.
Compound 4 (2.56g, 5.89mmol) is dissolved in 200mL 0.5M KOH/CH3OH solution.1mL β-sulfydryl is added
Acetic acid (14.4mmol).Reaction is stirred overnight under an argon atmosphere.After the reaction was completed, there is white heavy in the reactive mixture
It forms sediment.After filtering precipitating, methanol is carefully removed by Rotavap.120mL water is added into residue, and with 40mL EtOAc
Extraction is three times.Combined organic layer, which is saturated NaHCO3 solution with 120mL, washed once, and saline solution 120mL washed once.It will
Organic layer is dry with Na2SO4 and is removed by Rotavap, and obtaining 1.0g light yellow solid Compound 5, (5.4mmol, 68% produces
Rate).1H-NMR (CDCl3,400MHz) δ 7.22-7.30 (m, 4H), 4.29 (d, J=6.0Hz, 2H), 3.73 (s, 2H), 2.44
(s, 3H), 1.46 (s, 9H) .ESI-MS (M+H) +=251.17, Cal. (M+H) +=251.18.
Compound 5 (1.0 grams, 5.4mmol) is dissolved in 50mL DCM, is added acetic acid (0.324 gram, 5.4mmol), then
It is added EDC (1.15 grams, 6.0mmol), reaction is stirred overnight under argon atmosphere.After the reaction was completed, unsaturated carbonate hydrogen is first used
Sodium solution 50mL is washed twice, is then washed twice with 50mM hydrochloric acid solution 50mL.It after solvent seasoning, is removed, is obtained with rotary evaporation
1.43 grams of compound as white solid 6 (4.9mmol, 90% yield).1H-NMR(CDCl3,400MHz)δ 7.22-7.30(m,
4H), 4.29 (d, J=6.0Hz, 2H), 3.73 (s, 2H), 2.44 (s, 3H), 2.20 (s, 3H), 1.46 (s, 9H) .ESI-MS (M
+ H) +=293.10, Cal. (M+H) +=293.18.
Compound 6 (1.43 grams, 4.9mmol) is dissolved in 50mL 2M HCl/EtOAc, is stirred 2 hours, is occurred a large amount of white
Color precipitating, filter simultaneously washed with 20mL ethyl acetate, dry after obtain 0.89 gram of compound as white solid 7 (3.9mmol, 79%
Yield).1H-NMR (CDCl3,400MHz) δ 7.22-7.30 (m, 4H), 4.29 (d, J=6.0Hz, 2H), 3.73 (s, 2H),
2.44 (s, 3H), 2.20 (s, 3H) .ESI-MS (M+H) +=193.50, Cal. (M+H) +=193.13.
Mixed compound 7 (0.89g, 3.9mmol) and 1.1mL triethylamine in 30mL methylene chloride (DCM)
Then compound 2 (0.87 gram, 3.9mmol) is added in (7.8mmol).Reaction is stirred overnight under an argon atmosphere.Reaction is completed
Afterwards, 30mL DCM is added.Mixture is washed twice with 50mL HCl (50mmol/L) solution, 50mL is saturated NaHCO3 solution
It washes twice, and primary with 50mL aqueous salt solu-tion.It is removed organic layer anhydrous Na 2SO4 drying and by Rotavap,
Obtain 1.32 grams of faint yellow solid compounds 8 (3.5mmol, 90% yield).1H-NMR(CDCl3,400MHz)δ7.60-8.00
(m, 4H), 7.10-7.20 (m, 4H) 4.29 (d, J=6.0Hz, 2H), 3.73 (s, 2H), 2.44 (s, 3H), 2.20 (s, 3H)
.ESI-MS (M+H) +=378.45, Cal. (M+H) +=378.10.
Compound 8 (1.3g, 3.5mmol) is dissolved in 10mL DMF, and it is solid that 2.8g Na2CO3 (26.4mmol) is added
Body.Then 1.37mL methyl iodide (22mmol) is added.Reaction is stirred 2 hours in the dark.Check that reaction is completed by HPLC.
After the reaction was completed, major part CH 3I and DMF are removed by Rotavap.Residue is dissolved in 50mL EtOAc and 50mL salt water
In solution.Separate organic layer and with 60mL aqueous salt solu-tion three times, it is dry with Na 2SO 4 and by Rotavap removing, obtain
To 1.23 grams of yellowish solid chemical compounds 9 (3.15mmol, 90% yield).1H-NMR(CDCl3,400MHz)δ 7.60-8.00
(m, 4H), 7.10-7.20 (m, 4H) 4.29 (d, J=6.0Hz, 2H), 3.73 (s, 2H), 2.54 (s, 3H), 2.44 (s, 3H),
2.20 (s, 3H) .ESI-MS (M+H) +=392.45, Cal. (M+H) +=392.12.
Compound 9 (1.23g, 3.15mmol) is dissolved in 100mL 0.5M KOH/CH3OH solution.0.5mL β-is added
Thioacetic acid (7.2mmol).Reaction is stirred overnight under an argon atmosphere.After the reaction was completed, there is white in the reactive mixture
Precipitating.After filtering precipitating, methanol is carefully removed by Rotavap.120mL water is added into residue, and uses 40mL
EtOAc is extracted three times.Combined organic layer, which is saturated NaHCO3 solution with 60mL, washed once, saline solution 60mL washing one
It is secondary.It is removed organic layer Na2SO4 drying and by Rotavap, obtains 1.0 grams of light yellow solid Compounds 10
(2.05mmol, 65% yield) 1H-NMR (CDCl3,400MHz) δ 7.10-7.20 (m, 4H) 4.29 (d, J=6.0Hz, 2H),
3.73 (s, 2H), 2.80 (s, 3H), 2.44 (s, 3H), 2.20 (s, 3H) .ESI-MS (M+H) +=207.64, Cal. (M+H)+
=207.14.
0.41 g of compound 10 (2mmol) is dissolved in the methanol solution that 10mL contains 0.2mL acetic acid, 0.32 gram of chemical combination is added
Object 11 (2mmol), 310 milligrams of NaCNBH3 (5mmol) are dissolved in 10mL methanol, are slowly added into.After reaction stirring 2 hours, with rotation
Evaporimeter removes methanol.20mL semi-saturation sodium bicarbonate solution is added, is extracted with 20mL ethyl acetate organic after 3 merge
After mutually being washed with saturated salt, organic solvent is removed, uses methylene chloride and ethyl acetate as irrigation, it is pure on silica gel column chromatography
Change, obtains 0.594 gram of compound as white solid 12 (1.7mmol, 85% yield) 1H-NMR (CDCl3,400 MHz) δ 7.10-
7.20 (m, 4H) 4.29 (d, J=6.0Hz, 2H), 3.73 (s, 2H), 3.60 (m, 2H), 3.28 (m, 2H), 2.80 (s, 3H),
2.44 (s, 3H), 2.20 (s, 3H), 1.46 (s, 9H) .ESI-MS (M+H) +=350.44, Cal. (M+H) +=350.24.
Compound 12 (0.59g, 1.7mmol) is dissolved in 10mL DMF, and 1.4 grams of Na2CO3 (13.2mmol) are added
Solid.Then 0.7mL methyl iodide (12mmol) is added.Reaction is stirred 2 hours in the dark.It has been reacted by HPLC inspection
At.After the reaction was completed, most of iodomethane and DMF are removed by Rotavap.Solid purifies on silica gel column chromatography more than institute obtains
0.44 gram of compound as white solid 13 (1.2mmol, 70% yield) 1H-NMR (CDCl3,400MHz) δ 7.10-7.20 (m, 4H)
4.29 (d, J=6.0Hz, 2H), 3.73 (s, 2H), 3.60 (m, 2H), 3.28 (m, 2H), 2.82 (s, 3H), 2.80 (s, 3H),
2.44 (s, 3H), 2.20 (s, 3H), 1.46 (s, 9H) .ESI-MS (M+H) +=365.78, Cal. (M+H) +=365.26.
Compound 13 (0.44 gram, 1.2mmol) is dissolved in 50mL 2M HCl/EtOAc, is stirred 2 hours, is occurred a large amount of
White precipitate, filter simultaneously washed with 20mL ethyl acetate, dry after obtain 0.336 gram of compound as white solid 14 (1.0mmol,
85% yield) 1H-NMR (CDCl3,400MHz) δ 7.10-7.20 (m, 4H) 4.29 (d, J=6.0Hz, 2H), 3.73 (s, 2H),
3.60(m,2H),3.28(m,2H),2.82(s,3H),2.80(s,3H),2.44(s,3H),2.20 (s,3H).ESI-MS M+
=264.42, Cal.M+=264.21.
The QABIT of isotope labelling synthesis it is identical with the synthesis of unlabelled compound 14, but from synthesize compound
4, compound 6 uses following four groups of isotope labelling reactants during compound 13: (CH3I, CH3COOH,
13CHD2I)；(CH3I, CH313COOH, CHD2I)；(CHD2I, CH3COOH, 13CH3I)；(CHD2I, CH313COOH,
CH3I)。
Embodiment two: serum polysaccharide labeling method
The first step, the enrichment of N- polysaccharide.
First by 20 μ L haemocyanins in 200 μ L by 20 μ L denaturation buffers (10x) and 160 μ L buffer (pH 10.0；
40mmol/L sodium citrate and 20mmol/L sodium carbonate) composition solution in 10 minutes C are denaturalized at 100 DEG C.Slow with pH 10
After fliud flushing pre-processes Aminolink TM resin (200 μ L), denatured protein is added in 300 μ L buffers (pH 10.0)
Aminolink TM resin in, and at room temperature mixing be incubated for 4 hours.50 μ L500mmol/ L sodium cyanoborohydrides (1 are added
× PBS), then incubate 4 hours.After rinsing resin twice with 500 μ L1x PBS (pH 7.4), with the 50mmol/L in 1 × PBS
Sodium cyanoborohydride (NaCNBH3) goes back raw sample 4 hours.
It will be washed twice, remaining aldehyde with the pearl of protein-conjugate 1mol/L Tris-HCl (500 μ L, pH 7.6)
Site is with 500 μ L1mol/L Tris-HCl in 50mmol/L NaCNBH3 (0.5 hour).1mol/L of 400 μ L of pearl
NaCl is washed three times, is washed three times with H2O.In order to stablize sialic acid residues, by the polysaccharide and 465 μ L couple on solid support
Toluidines (Sigma) solution (pH 4-6) is incubated for, by 400 μ L para-totuidine, 25 μ L36-38%HCl and 40 μ L EDC (N-
(3- dimethylaminopropyl)-N'- ethyl carbodiimide；5.6mol/L；Sigma).Progress 3 hours at room temperature are reacted, so
Afterwards with 1% formic acid (twice) of 400 μ L, 10% acetonitrile (twice) of 400 μ L, the 1mol/L NaCl (2 times) of 400 μ L is twice),
It is finally H2O (twice).With by 4 μ LPNGase F (New England BioLabs), 16 μ L10 × G7 buffers, 146 μ L
160 μ l solution of water composition discharge N- polysaccharide, are incubated at least 2 hours at 37 DEG C.Before vacuum drying, pass through Carbograph
The polysaccharide of TM purifying elution.Yang, S.J.；Zhang, H., Anal.Chem.2012,84,2232-2238.
Step 2: N-glvcan is marked.
In the presence of 1mol/L NaCNBH 3, dry serum polysaccharide is resuspended in 80 μ L by dimethyl sulfoxide (DMSO)
In the solution mixture of acetic acid (AA) (7:3, volume) composition.By sample with the ratio of 1:1:2:4 (or 10:10:20:40 μ L)
Example is divided into four bottles.To be dissolved in the mixture of DMSO and AA (7:3) 40 μ L100mmol/L QABIT (176,177,
It 178 and 179) is separately added into each sample, and is incubated 4 hours at 65 DEG C.It is quenched by the way that 2mL water and the 2 dense formic acid of μ L is added
Reaction.
It is used to clean by Carbograph TM by being collected with the sample of 4 weight QABIT labels.The sample of purifying is dissolved
In 0.2% formic acid of 400 μ L.
Step 3: MS/MS analysis method and parameter.
In the unlimited HPLC- chip cube nano-spray of Agilent 1260 being connect with Agilent 6550Q-TOF MS
LC-MS experiment is carried out on device.HPLC- chip is enriched with trapping column and 75 μ m 150mm analytical column (5 μm, 300SB) groups by 160nL
At.Mobile phase is: the acetonitrile of the nanopure water of (A) with 0.1% formic acid and (B) with 0.1% formic acid.Using 65 minutes long
Gradient method carries out LC separation.The sample loading on enriching column is carried out at 1%B.Gradient for analytical column starts from 1%
B rose to 40%B at 35 minutes, rose to 85%B at 42 minutes, maintained 85%B up to 48 minutes, then at 55 minutes
When return to 1%B, and run forward horizontal stand 10 minutes next time.With the flow velocity load sample of 4 μ L/min, and with 0.35 μ L/min
Elution.Q-TOF is operated under high-resolution positive ion mode.Crucial MS parameter is: 225 DEG C of source temperature, capillary voltage
1900V is crushed voltage 155V, dry gas flow velocity 13L/min.With 4 spectrum/second under MS mode between m/z 100-3000
Sweep speed and MS/MS mode under 5 spectrum/second obtain data.4 are set as with slope, offset is set as 3 for double charge
Ion applies slope collision energy；Slope is 2, for three times or higher change ion, deviating is 2.Use Agilent
MassHunter data acquisition software realizes system control, and carries out data with MassHunter qualitative analysis (B.06.00)
Analysis.Glycosyl identification is carried out using GlycoWorkbench software (version 2 .0).The representative MS2 spectrum of the N- polysaccharide of label
It is provided in Fig. 4-Figure 13.These large fragments spectrally are useful, and indicated by an arrow for polysaccharide structures identification
Reporter ion (176,177,178 and 179) is quantitative for polysaccharide.
The data of three: QABIT label of embodiment are analyzed.
As other isobar labels for peptide and small molecule, the 4- weight QABIT reagent of test be have it is identical
One group of four molecule of chemical structure and molecular weight, but they contain different stable isotope core, such as 13C and 2H.They
Structure by from 176 to 179 dalton of molecular weight ranges series report molecule, compensation report molecular mass difference counterbalance and
It is made up of the reactive primary amine of reduction amination and polysaccharide coupling.The label chemistry is similar to by 2-AA/2-AB (2- aminobenzoic
Sour (2-AA) or 2- aminobenzamide (2-AB)), Bigge, JC et al. " Nonselective and efficient
Fluorescent labeling uses 2- aminobenzamide and ortho-aminobenzoic acid, Anal.Biochem.230,229-238
(1995)), therefore the well known scheme for 2-AA/2-AB label can be modified in this in the case where no many changes
In field.
However, the significant difference between label and 2-AA/2-AB of the invention be hydrone naturally and stoichiometrically
It is lost from the polysaccharide that QABIT is marked, and only the polysaccharide of 2-AA/2-AB label shows the partial loss of hydrone.This phenomenon
(i.e. neighbouring to participate in reaction) can be formed by hexatomic ring advantageous on energy to carry out.Cai,Y.,Ling,C.-C.&Bundle,
D.R."Facile approach to 2-acetamido-2-deoxy-β-D-glucopyranosides via a
furanosyl oxazoline"Org.Lett.7,4021-4024(2005).In MS2 fragmentation, the polysaccharide of QABIT label is produced
Raw strong report ion is used for accurate quantification, without additional cation such as Na+ or metal ion, to eliminate ion
Depression effect simultaneously prevents from forming multiple H+/Na+ adducts.In addition, cooperation has the polysaccharide of permanent positively charged quaternary amine can be with
Enhance their ionization in MS.Therefore, the significant enhancing of the detection sensitivity of polysaccharide, this is analyzing low abundance polysaccharide or finite quantity
Sample when be advantageous.
In order to which application QABIT carries out Analysis of polysaccharides, the scheme based on solid phase is developed, as shown in Figure 2.Yang,S.,Li,
Y., Shah,P.&Zhang,H.,"Glycomic analysis using glycoprotein immobilization for
glycan extraction"Anal.Chem.85,5555-5561(2013).Glycoprotein is firstly fixed on pearl, and in carbon two
It is handled in the presence of imines coupling agent with excessive para-totuidine.Saliva can be conjugated with para-totuidine on polysaccharide in the step completely
Acid, to stablize these unstable residues during MS is analyzed.Then N- is discharged from the glycoprotein of immobilization with PNGase F
Polysaccharide leads to the quaternary amine for being used to mark containing isotopic tag reagent in the aldehyde radical of reduction end exposure.Then reverse phase liquid is used
Chromatography (RPLC) is coupled the polysaccharide of tandem MS analysis label.Para-totuidine moiety hydrophobic in this way is to each sialic acid, institute
It can easily be solved on a cl 8 column with carrying the N- polysaccharide of different number of sialic acid.
Single isotopic mass M=FaNbHcSdGe of the polysaccharide of label is calculated based on equation 2.
M=C+a × F+b × N+c × H+d × S+e × G+ (d+e) × pT+Q (2).
Wherein: C represents N- polysaccharide nuclear structure, 910.3278Da；F is fucose, 146.0579Da；N is HexNAc,
203.0794Da；H is hexose, 162.0528Da；S is Neu5Ac, 291.0954Da；G is Neu5Gc, 307.0903Da；PT is
Para-totuidine, 89.0629Da (lose a kind of water after) are coupled in scheme with each sialic acid；Q is QABIT,
233.2147Da (losing an oxygen due to reduction amination and losing a water since adjacent participation is reacted)；A, b, c,
D, e are the numbers of each corresponding units.Formula of the nuclear structure (2HexNAc and 3 hexoses) from the N- polysaccharide of this paper
(FaNbHcSdGe) it is excluded in.B and c respectively indicate HexNAc other than HexNAc the and Hexose unit in nuclear structure and
Hexose unit.
In ESI, the polysaccharide of label is generally detected as to carry the ion of multiple charge (z), and the m/z observed is B.
We can calculate its single isotopic mass (M') based on equation 3.
Equation 3:M'=B × Z+ (Z-1.0078)
In order to identify the polysaccharide from MS experiment, the N- polysaccharide of all QABIT labels in polysaccharide library can be calculated first
Single isotopic mass (M), such as the alliance for functional sugar group (CFG) database 17.Then, single isotope calculates MS1
The quality (M') of spectrum intermediate ion, and match with the single isotopic mass (M) calculated from polysaccharide database to find that it is formed.
Example IV: QABIT label is compared with city pin aminoxyTMT is for polysaccharide researches.
According to saying description method in example two, in 1% standard sialoglycopeptide (SGP), two kinds of N- polysaccharide of extraction
(N2H2S2>90% and N2H2S<10%) is marked with QABIT.As a comparison, SGP polysaccharide is based on uncle also by commercially available
The aminoxyTMT of the polysaccharide of amine is marked.
Figure 14 shows that unlabelled SGP polysaccharide, Figure 15 show that the SGP polysaccharide of aminoxyTMT label, Figure 16 are shown
The MS map of the polysaccharide of QABIT label.
The polysaccharide of QABIT label only show two labeled as N2H2S and N2H2S2 it is unimodal, show label reaction completion.
Although aminxyTMT label polysaccharide also show two main peaks, we still observe unlabelled N2H2S and
N2H2S2, shows to react with the label of aminoxyTMT and is more difficult to complete.In addition, the polysaccharide of aminoxyTMT label shows have
Multiple satellites of 14 dalton mass difference, keep MS1 more complicated and equalize peak intensity.The experiment clearly demonstrates QABIT
The completion of label and its exceed existing commercial reagent aminoxyTMT the advantages of.
Embodiment five: QABIT quantifies the research of polysaccharide.
According to description method is said in example two, by marking the tire ball egg from tire ox with 4 weight QABIT of 1:1:3:5 ratio
White (200 μ g, 200 μ g, 600 μ g and 1000 μ g) polysaccharide tests the quantitative precision (Figure 17-Figure 19) of QABIT.Figure 17 is aobvious
The MS1 of the myosin N- polysaccharide of QABIT label, including N2H2S2, N3H3S2, N3H3S3 and N3H3S4 are shown.Figure 18 is generation
The MS/MS map of table polysaccharide N2H2S2 comprising a series of polysaccharide fragments and range from 176 to 179 strong report son from
Son.Figure 19 shows the relative abundance of the representative polysaccharide N2H2S2 from four primary samples.The experimental result of the polysaccharide is very
Close to 1:1:3:5.The linear dependence between theoretical ratio of measurement and from independent duplicate small standard deviation three times
Show the quantitative fabulous reproducibility of QABIT.
The technical means disclosed in the embodiments of the present invention is not limited only to technological means disclosed in above embodiment, further includes
Technical solution consisting of any combination of the above technical features.It should be pointed out that for those skilled in the art
For, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also considered as
Protection scope of the present invention.
Claims (10)
1. a kind of N- Analysis of polysaccharides method, it is characterised in that: with comprising MS/MS can shear key and can with N- polysaccharide be conjugated it is anti-
The isotopic tag reagent containing quaternary amine of answering property group marks N- polysaccharide.
2. N- Analysis of polysaccharides method as described in claim 1, it is characterised in that: the isotopic tag reagent containing quaternary amine
Including following formula: report group-balance group-reaction group, wherein reporter group and balance group, which pass through MS/MS, can shear key connection.
3. N- Analysis of polysaccharides method as claimed in claim 2, it is characterised in that: the isotopic tag reagent containing quaternary amine
Including formula: reporter group-balance group-reactive group, specific structure are
Wherein at least one of position 1-3 includes that isotope atom, reporter group and balance group can shear keys by MS/MS
Connection, and R is the reactive group that can be conjugated with polysaccharide.
4. N- Analysis of polysaccharides method as claimed in claim 3, it is characterised in that: the quality of the reporter group 176 to
In the range of 179Da, and the reagent contains 2 or 3 isotope atoms independently selected from 13C and 2H.
5. N- Analysis of polysaccharides method as described in claim 1, it is characterised in that: the N- polysaccharide is prepared by the following:
(1) the fixed glycoprotein comprising N- polysaccharide on solid support；
(2) N- polysaccharide described in chemical modification；
(3) N- polysaccharide is discharged from solid support；
Wherein the glycoprotein is obtained from people or non-human animal's serum；
The N- polysaccharide carries sialic acid, and the chemical modification of the N- polysaccharide includes the carbodiimide coupling of sialic acid；
To N- polysaccharide quantitative analysis, the quantitative analysis step includes fragmentation, quantitative two steps, and the N- polysaccharide after label is in MS/
Fragments characteristic peak is generated in MS, and N- polysaccharide is quantified according to the intensity of characteristic peak；
Wherein Na+It is not used in the N- polysaccharide of quantitative analysis label.
6. a kind of isotopic tag reagent containing quaternary amine, it is characterised in that: under the isotopic tag reagent containing quaternary amine includes
Formula: reporter group-balance group-reactive group,
Specific structure is
Wherein at least one of position 1-3 includes isotope atom, and reporter group and balance group pass through MS/MS scissionable bond
Connection, and R is the reactive group that can be conjugated with polysaccharide.
7. the isotopic tag reagent containing quaternary amine as claimed in claim 11, it is characterised in that: the isotope containing quaternary amine
Tagging reagents are the isobar tagging reagents containing quaternary amine, and R is the one of which of primary amine, hydrazides or aminooxy group.
8. the isotopic tag reagent containing quaternary amine as claimed in claim 11, it is characterised in that: the wherein reporter group
Quality in the range of 176 to 179Da, the reagent contain independently selected from13C and22 or 3 isotope atoms of H, packet
Containing the reactive primary amine that can pass through reduction amination with polysaccharide conjugation.
9. the isotopic tag reagent containing quaternary amine as claimed in claim 11, it is characterised in that: wherein described to contain same amount
The quaternary amine of different sequence label has structure (I):
And wherein 3 methyl are13CHD2；
Wherein the quaternary amine containing isobaric label has structure (I):
And wherein 1 carbon is13C, 3 methyl are CHD2；
Wherein the quaternary amine containing isobaric label has structure (I):
And wherein 2 methyl are CHD2, 3 methyl are13CH3；
Wherein the quaternary amine containing the different sequence label of same amount has structure (I):
And wherein 1 carbon is13C, 2 methyl are CHD2。
10. a kind of isotopic tag Reagent Protocol being synthetically prepared containing quaternary amine, it is characterised in that: the following steps are included:
(a) it reacts compound 1 with compound 2, obtains compound 3；
(b) make compound 3 and sodium carbonate and methyl Iod R, obtain compound 4；
(c) make compound 4 and β-thioacetic acid, potassium hydroxide solution reaction obtains compound 5；
(d) make compound 5 and acetic acidreaction, obtain compound 6；
(e) tertbutyloxycarbonyl (Boc) protecting group is removed using the ethyl acetate solvent of hydrogen chloride in compound 6, obtains chemical combination
Object 7；
(f) it reacts compound 7 with o- nitrobenzene sulfonyl chloride, obtains compound 8；
(g) make compound 8 and sodium carbonate and methyl Iod R, obtain compound 9；
(h) make compound 9 and β-thioacetic acid, potassium hydroxide solution reaction obtains compound 10；
(i) it reacts compound 10 with tertbutyloxycarbonyl -2- aminoacetaldehyde (reactant 11), obtains compound 12；
(j) make compound 12 and sodium carbonate and methyl Iod R, obtain compound 13；
(k) tertbutyloxycarbonyl (Boc) protecting group is removed using the ethyl acetate solvent of hydrogen chloride in compound 13, obtained final
Product compound 14；
Synthesis technology is as follows:
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