CN112778194A - Universal low-cost quaternary ammonium salt sugar chain isotope labeling reagent and synthetic method thereof - Google Patents
Universal low-cost quaternary ammonium salt sugar chain isotope labeling reagent and synthetic method thereof Download PDFInfo
- Publication number
- CN112778194A CN112778194A CN202011553376.7A CN202011553376A CN112778194A CN 112778194 A CN112778194 A CN 112778194A CN 202011553376 A CN202011553376 A CN 202011553376A CN 112778194 A CN112778194 A CN 112778194A
- Authority
- CN
- China
- Prior art keywords
- sugar chain
- labeling reagent
- isotope labeling
- cost
- quaternary ammonium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/06—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom containing only hydrogen and carbon atoms in addition to the ring nitrogen atom
- C07D213/16—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom containing only hydrogen and carbon atoms in addition to the ring nitrogen atom containing only one pyridine ring
- C07D213/20—Quaternary compounds thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/06—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom containing only hydrogen and carbon atoms in addition to the ring nitrogen atom
- C07D213/127—Preparation from compounds containing pyridine rings
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
- G01N27/626—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode using heat to ionise a gas
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/05—Isotopically modified compounds, e.g. labelled
Abstract
The invention relates to the technical field of bioglycomics analysis, and discloses a general low-cost quaternary ammonium salt sugar chain isotope labeling reagent and a synthesis method, wherein the general low-cost quaternary ammonium salt sugar chain isotope labeling reagent comprises a heavy isotope labeling reagent deuterated 7-4-methyl-1- (2-hydrazino-2-oxoethyl) -pyridinium bromide and a corresponding light isotope labeling reagent 4-methyl-1- (2-hydrazino-2-oxoethyl) -pyridinium bromide; the method can be used for mass spectrum high-sensitivity and high-accuracy relative quantitative analysis of biological reducing sugar chains with different molecular weight segments; the reagent provided by the invention can greatly improve the mass spectrum detection sensitivity of the sugar chain; simultaneously, the method is suitable for the mass spectrum relative quantitative analysis of the reducing sugar chains with smaller and larger molecular weights; the cost of the synthetic raw materials is low, and the defect that the cost of the hydrazide-based quaternary ammonium salt stable isotope labeling reagent synthesized based on the deuterated isoquinoline is high is overcome.
Description
Technical Field
The invention relates to the technical field of analysis of biological sugar components, in particular to a general low-cost quaternary ammonium salt sugar chain isotope labeling reagent and a synthesis method thereof.
Background
Glycans are a class of important biomolecules that are present in a wide variety of organisms, including polysaccharides, free oligosaccharides, and glycoconjugate sugar chains. These polysaccharides play an important role in various vital activities, for example, some non-digestible plant polysaccharides and animal breast milk oligosaccharides can promote the proliferation of beneficial intestinal flora, thereby playing a series of physiological regulation roles. Glycoproteins are important glycoconjugates, and O-linked sugar chains on serine or threonine residues (-Ser/Thr-) and N-linked sugar chains on asparagine residues (-Asn-) of the glycoconjugates, which are used as biological information molecules, play important roles in various biological processes such as cell recognition, cell adhesion, signal transduction, immune response and the like, and various diseases such as pathogen infection, glycosylation deficiency syndrome, tumors and the like are closely related to glycoprotein sugar chains. Human milk, eggs, and other foods contain abundant glycoprotein components, and sugar chains on these glycoproteins have important effects on promoting human health, for example, fucosylated sugar chains have an immunomodulatory effect, while sialylated sugar chains promote brain development in newborn infants. The structure and amount of sugar chains in biological tissues are closely related to their biological functions, and the kind and amount of sugar chain components in food also have an important influence on their biological effects. Therefore, structural analysis and quantitative analysis of sugar chains are important for screening disease sugar chain markers and for development and utilization of natural sugar chain resources.
The structure analysis and quantitative analysis of sugar chains are mainly performed by Mass Spectrometry (MS). Among sugar chain structure analyses, tandem mass spectrometry (MS/MS) is the most commonly used means, and allows fragmentation (fragmentation) of a fully methylated modified (Per-methylation) sugar chain, and the monosaccharide sequence and the glycosidic bond site of the sugar chain can be determined by analyzing the sugar chain parent ion cleavage mechanism and assigning the structure of the sugar chain fragment ion. The sugar chain quantification methods mainly include High Performance Liquid Chromatography (HPLC), high performance liquid chromatography-mass spectrometry (LC-MS) and MS. The HPLC method can quantify the target sugar chain by the chromatographic peak area. The LC-MS method not only can realize the HPLC separation of sugar chains with different structures, but also can carry out online sequence identification through MS and MS/MS and carry out quantitative analysis through chromatographic peak areas. The MS method is characterized in that two sugar chain samples derived from an equivalent biological sample are subjected to derivatization labeling in a light isotope form and a heavy isotope form of the same labeling reagent respectively, the obtained samples are mixed and used for mass spectrometry detection, a sugar chain sequence can be identified through MS and MS/MS analysis, the content ratio of each sugar chain in the two samples can be determined through the mass spectrum signal intensity ratio of a light isotope label and a heavy isotope label of each sugar chain, and the analysis strategy is called mass spectrum relative quantification. Among these methods, mass spectrometry has the advantages of simple operation, sensitive detection, accurate result, and the like, and is the most commonly used method for quantitative glycomics analysis.
At present, the types of stable isotope labeling reagents used for the relative quantification of sugar chain mass spectrometry are more, including13C6-2-aminobenzoic acid (f)13C6-2-AA)、d6-2-aminopyridine (d)6-2-AP)、13C6-aniline (b)13C6-An)、d5-1-phenyl-3-methyl-5-pyrazolone (d)5-PMP)、13C6-4-phenethyl-benzoic hydrazide (13C6-4-PEBH), and the like. Among these reagents, quaternary ammonium salt reagents having an active hydrazide group have advantages over other reagents in terms of improvement of detection sensitivity of sugar chain mass spectrometry and simplification of detection signals of sugar chain mass spectrometry, and thus have been increasingly emphasized in sugar chain mass spectrometry versus quantitative analysis. Hitherto, stable isotope labeling agents of hydrazidyl quaternary ammonium salts have been reported to include deuterated Girard reagent P (d)5-GP) and deuterated 2- (2-hydrazine-2-ethoxy) isoquinoline-2-ammonium bromide (d)7-HIQB). In the mass spectrometric relative quantitative analysis of sugar chains, d of each sugar chain5-GP derivatives with corresponding light isotope reagents GP (d)0GP) derivative has a mass difference of 5Da between mass spectrum signal peaks, and can carry out rapid, sensitive and accurate relative quantitative analysis on reducing sugar chains with relatively small molecular weight, but the reducing sugar chains with relatively large molecular weight have d0-GP derivatives and corresponding d5The large overlap between the peaks of the mass spectrum signals of the GP derivatives resultsThe accuracy of the quantitative result is greatly reduced. d7The HIQB marker can then overcome d5The deficiency of the GP labeling method, because of the d of each sugar chain7HIQB derivatives with the corresponding light isotope reagent HIQB (d)0HIQB) derivative, and mass difference of 7Da exists between mass spectrum signal peaks, so that mass spectrum quantitative analysis of reducing biological sugar chains with different molecular weight segments can be basically and simultaneously satisfied. However, d7HIQB is difficult to be widely applied due to the fact that synthetic raw materials are expensive and the use economy is poor.
Therefore, how to overcome d7The HIQB has the defects that the development cost is lower, and the hydrazide quaternary ammonium salt stable isotope labeling reagent is suitable for mass spectrum high-sensitivity quantitative analysis of bioreductive sugar chains with different molecular weight segments, and becomes a technical problem to be solved urgently in glycomics analysis.
Disclosure of Invention
In order to solve the defects of the prior art, the general low-cost quaternary ammonium salt sugar chain isotope labeling reagent and the synthesis method provided by the invention have the advantages of strong universality, low cost, high sensitivity and accurate quantification.
The invention provides a general low-cost quaternary ammonium salt sugar chain stable isotope labeling reagent, which comprises a heavy isotope labeling reagent and a corresponding light isotope labeling reagent, wherein the heavy isotope labeling reagent has a structural formula shown in formula (I):
the structural formula of the labeling reagent in the form of the light isotope is shown as a formula (II):
the second object of the present invention is to provide a method for synthesizing the above general low-cost reagent for labeling sugar chains with stable isotopes of quaternary ammonium salts, which comprises the steps of:
s1, taking deuterated 4-methylpyridine or 4-methylpyridine and ethyl bromoacetate as raw materials, and generating a mixture containing a compound shown in a formula (III) through a nucleophilic substitution reaction between amine and alkyl halide at 65-70 ℃;
s2, adding hydrazine hydrate into the mixture S1 in an ice bath environment, and preparing deuterated 7-4-methyl-1- (2-hydrazino-2-oxoethyl) -pyridinium bromide or 4-methyl-1- (2-hydrazino-2-oxoethyl) -pyridinium bromide through nucleophilic substitution reaction between ester and amine;
the synthetic route is as follows:
preferably, in S1, the solvent is methanol or ethanol.
Preferably, in S1, ethyl bromoacetate: the molar ratio of the deuterated 4-methylpyridine or the 4-methylpyridine is 1.1-1.5: 1.
Preferably, in S1, the reaction time is 12 h.
Preferably, in S2, hydrazine hydrate is dripped into the S1 mixture under stirring, and after dripping is completed within 30min, the reaction is continued for 2h in an ice bath environment.
Preferably, in S2, hydrazine hydrate: the molar ratio of ethyl bromoacetate is 1.1-1.5: 1.
Preferably, in S2, after the reaction is finished, the product is obtained by concentrating, then precipitating with absolute ethyl alcohol precooled at the temperature of minus 20 ℃, filtering, washing and drying.
The third purpose of the invention is to provide the application of the general low-cost quaternary ammonium salt type sugar chain stable isotope labeling reagent in the aspect of the mass spectrum relative quantitative analysis of the biological reducing sugar chain, which comprises the following steps:
two reducing sugar chain samples derived from an equivalent biological sample are respectively subjected to hydrazone reaction with 4-methyl-1- (2-hydrazino-2-oxoethyl) -pyridinium bromide and deuterium 7-4-methyl-1- (2-hydrazino-2-oxoethyl) -pyridinium bromide under the condition that the pH value is 2-5, a first mixture and a second mixture are prepared, the first mixture and the second mixture are mixed, an aqueous solution is prepared after concentration, and detection and analysis are carried out through mass spectrometry.
Preferably, the mass spectrum is electrospray ionization mass spectrum or matrix-assisted laser desorption ionization-time-of-flight mass spectrum.
Compared with the prior art, the invention has the following beneficial effects:
(1) the stable isotope reagent deuterium 7-4-methyl-1- (2-hydrazino-2-oxoethyl) -pyridinium bromide and 4-methyl-1- (2-hydrazino-2-oxoethyl) -pyridinium bromide provided by the invention are quaternary ammonium compounds with hydrazide groups, can be labeled at the reducing end of a biological sugar chain under the mild weak acid condition, and the positive charge of the compound can simplify the peak form and greatly improve the mass spectrum detection sensitivity in the mass spectrum analysis of the reducing sugar chain;
(2) the stable isotope reagents deuterium 7-4-methyl-1- (2-hydrazino-2-oxoethyl) -pyridinium and 4-methyl-1- (2-hydrazino-2-oxoethyl) -pyridinium have the relative molecular mass difference of 7Da, and the relative molecular masses of the reagents are different from each other by 7Da, so that the mass difference of 7Da can be formed by matching the reagents, the reagents have good resolution in the mass spectrum relative quantitative analysis of the bioreductive sugar chains with molecular mass sections above 2000Da, and the reagents are also suitable for molecular mass sections below 2000 Da;
(3) the synthetic raw material deuterium 7-4-methylpyridine of the stable isotope reagent deuterium 7-4-methyl-1- (2-hydrazino-2-oxoethyl) -pyridinium provided by the invention is a commercial reagent, compared with d7Starting materials d for the synthesis of HIQB7-isoquinoline, d7The price of the-4 methylpyridine is cheaper, the development cost is lower, and the wide application can be realized.
Drawings
FIG. 1 shows the reagent d of the present invention0-HMP structural characterization includes1HNMR and13CNMR；
FIG. 2 shows a stable isotope reagent d of the present invention7-HMP structural characterization includes1HNMR and13CNMR；
FIG. 3 is a MALDI-MS spectrum of a mixture of marking reagents HMP and GP reagents and GT reagent derived maltohexaose and beta-cyclodextrin according to the present invention;
FIG. 4 is a linear investigation of the relative quantification of mass spectra of the labeled reagent of the present invention;
wherein 4A is d0-HMP-labelled maltodextrin and d7MALDI-TOF MS spectrum of an equal proportion mixture of HMP-labeled maltodextrins, FIG. 4B for the stable isotope reagent d0/d7Linear range investigation of HMP in relative quantification of sugar chains;
FIG. 5 shows a stable isotope labeling reagent d of the present invention0/d7-mass spectrum comparison of the products of labelling maltodextrin with HMP and with the traditional stable isotope reagent d0/d5-GP, respectively;
FIG. 6 is a MALDI-TOF MS spectrum of chicken albumin N-sugar chain;
wherein 6A is MALDI-TOF MS spectrum of underivatized labeled N-sugar chain, and each sugar chain is represented by [ M + Na ]]+And [ M + K]+Presenting the form; FIG. 6B is a MALDI-TOF MS spectrum of chicken albumin N-sugar chains labeled with HMP as a labeling reagent of the present invention, each sugar chain being represented by [ M ]]+Presenting the form;
FIG. 7 is a MALDI-TOF MS spectrum of a product obtained after N-sugar chains released from fetal bovine serum albumin are specifically amidated and modified by a sialic acid linkage and derivatized and labeled by a reducing-end HMP;
FIG. 8 shows d of highly fucosylated N-sugar chains released from human sperm protein0-HMP and d7-MALDI-TOF MS spectrum of an equal proportion mixture of HMP derivatives;
FIG. 9 is a MALDI-TOF MS spectrum of N-sugar chains of pig serum and chicken serum;
wherein, FIG. 9A is d of a product of specific amidation modification of N-sugar chain released from chicken serum protein by sialic acid linkage0-MALDI-TOF MS spectrum of HMP derivatives; FIG. 9B is d of a product of specific amidation of N-sugar chain released from porcine serum protein by sialic acid linkage7-MALDI-TOF MS spectrum of HMP derivatives; FIG. 9C is a MALDI-TOF MS spectrum of an equal proportion mixture of the samples of FIGS. 9A and 9B;
in FIGS. 6, 7, 8 and 9, the gray circles represent mannose, the black squares represent N-acetylglucosamine, the black triangles represent fucose, the white circles represent galactose, the black diamonds represent N-acetylneuraminic acid, and the gray diamonds represent N-glycolylneuraminic acid.
Detailed Description
The following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers. The examples described below, unless otherwise indicated, all temperatures are given in degrees Celsius, the reaction temperature is room temperature, room temperature means 25 ℃. + -. 5 ℃ and all temperature errors are. + -. 5 ℃.
In the following examples, quinoline, 4-methylpyridine, deuterated 4-methylpyridine, absolute ethanol, 80% hydrazine hydrate, Fetal Bovine Serum (FBS), and maltodextrin were obtained from Sigma, USA; PNGase F available from NewEngland BioLabs; sodium Dodecyl Sulfate (SDS), Dithiothreitol (DTT), NP-40, alpha 2.3-sialyllactose, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC.HCl), isopropylamine hydrochloride (iPrNH)2HCl), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), N-methylmorpholine, methylamine hydrochloride, ethyl bromoacetate are products of shanghai alatin corporation; c18 solid phase extraction column was purchased from Waters, porous graphite carbon column was purchased from Agela technologies; double distilled water is prepared by a laboratory double pure water distiller; other reagents are all domestic analytical purifiers.
In the present invention, a matrix-assisted laser ionization time-of-flight mass spectrometer (MALDI-TOF MS, Shimadu, Japan) was used for mass spectrometry. MALDI-TOF MS parameters were set as follows: the detection mode is a reflection mode; the mass-to-charge ratio (m/z) range is 600-6000, the detection energy is 100-119mV, the light source is nitrogen laser 355nm, the frequency is 40.0Hz, the acceleration voltage is 20kv, the laser is accumulatively irradiated for 1000 times at 2 positions of the target spot, and DHB is used as a laser auxiliary matrix during detection; the loading format was a sandwich format, i.e., 0.5. mu.L of DHB (20mg DHB in 0.5mL 50% acetonitrile in 0.05% TFA), 0.5. mu.L of sample, 0.5. mu.L LDHB were loaded onto the target plate, air dried and subjected to MALDI-TOF MS detection.
The Chinese corresponding to the abbreviations in the present invention is as follows:
ACN (acetonitrile), arb (arbitraryunit, arbitrary unit, belonging to pressure unit), DMSO (dimethyl sulfoxide), DTT (dithiothreitol), FBS (fetal bovine serum), MeOH (methanol), MS (bovine serum albumin), and so onn(Multi-stage Mass Spectrometry), mL (mL), min (min), ms (ms), h (hr), Relative abundance, SDS (sodium dodecyl sulfate), kV (kilovolt), V (volt), M (mol/L), TFA (trifluoroacetic acid), d (Mol/L)0HMP or HMP (4-ethyl-1- (2-hydrazino-2-oxoethyl) -pyridinium Bromide), d7-HMP (deuterium 7-4-methyl-1- (2-hydrazino-2-oxoethyl) -pyridinium bromide), GP (gilrad reagent P), GT (gilrad reagent T).
The invention provides a universal low-cost quaternary ammonium salt sugar chain stable isotope labeling reagent, which comprises a heavy isotope labeling reagent and a corresponding light isotope labeling reagent, wherein the structural formula of the heavy isotope labeling reagent is shown as the formula (I):
the structural formula of the labeling reagent in the form of the light isotope is shown as a formula (II):
the application of the general low-cost quaternary ammonium salt sugar chain stable isotope labeling reagent in the aspect of the relative quantitative analysis of the biological reducing sugar chain mass spectrum comprises the following steps:
two reducing sugar chain samples derived from an equivalent biological sample are respectively subjected to hydrazone reaction with 4-methyl-1- (2-hydrazino-2-oxoethyl) -pyridinium bromide and deuterium 7-4-methyl-1- (2-hydrazino-2-oxoethyl) -pyridinium bromide under the condition that the pH value is 2-5, a first mixture and a second mixture are prepared, the first mixture and the second mixture are mixed, an aqueous solution is prepared after concentration, and detection and analysis are carried out by mass spectrum, wherein the reaction mechanism is as follows:
the mass spectrum is electrospray ionization mass spectrum or matrix-assisted laser desorption ionization-time-of-flight mass spectrum.
Several examples are set forth below to illustrate in detail the reagents of the invention and their methods of synthesis and performance.
Example 1
A general low-cost synthesis method of quaternary ammonium salt sugar chain isotope labeling reagent comprises the following steps:
s1, a 25mL specification round bottom flask containing 10mL of methanol is placed on an electronic balance, 1.0g of deuterated 4-methylpyridine is weighed into the round bottom flask, and 2.0g of ethyl bromoacetate is weighed into the round bottom flask. A condensation reflux device is set up, and stirring reflux is carried out for 12 hours at 70 ℃;
s2, placing the obtained mixture in an ice bath, dropwise adding 1.2mL of 80% hydrazine hydrate under stirring, controlling the flow rate to be completed within 30min, sealing the round-bottom flask, and continuously stirring and reacting in the ice bath for 2 h;
s3, after the reaction is finished, concentrating the reaction liquid to be oily by using a rotary evaporator, then adding 10mL of anhydrous ethanol pre-cooled at-20 ℃, stirring in an ice bath for 30min to generate white precipitate, then performing suction filtration to obtain a white solid deuterium 7-4-methyl-1- (2-hydrazino-2-oxoethyl) -pyridinium bromide, washing the white solid for 3 times by using 2-3mL of anhydrous ethanol pre-cooled in advance, and finally completely drying the solid by using a vacuum drying oven;
s4, synthesizing 4-methyl-1- (2-hydrazino-2-oxoethyl) -pyridinium bromide by using 4-methylpyridine as a substrate according to the method.
5mg of the white solid obtained from S3 and S4 were dissolved in 1mL of D2In O, then carrying out1HNMR and13and (5) carrying out CNMR detection. As shown in FIG. 1 and FIG. 2, the results show that the obtained product conforms to the characteristic signal peaks of deuterium 7-4-methyl-1- (2-hydrazino-2-oxoethyl) -pyridinium bromide and 4-methyl-1- (2-hydrazino-2-oxoethyl) -pyridinium bromide, and the accuracy of the product structure is proved.
Example 2
The mass spectrometric detection sensitivity evaluation of the sugar chain derivative of the general low-cost quaternary ammonium salt sugar chain isotope labeling reagent comprises the following specific operation steps:
s1, weighing 9.9mg of maltohexaose and 11.34mg of beta-cyclodextrin, preparing into 1mmol/L aqueous solution respectively, mixing the maltohexaose and the beta-cyclodextrin solution according to the molar ratio of 5:1, and analyzing the obtained mixture solution by MALDI-TOF MS for later use.
S2, 24.5mg of HMP was dissolved in 1mL of a solvent (methanol: water: glacial acetic acid: 6:3:1) to prepare a 0.1M HMP reaction solution.
S3, S1, adding 25uL of HMP reaction solution, reacting in a water bath at 70 ℃ for 1h, centrifugally concentrating to remove the solvent, dissolving with 1mL of ultrapure water, and using for MALDI-TOF MS detection.
Comparative example 2-1
GT and GP are adopted as labeling reagents, the labeling objects are the same as the labeling objects in the embodiment 2, and are mixtures with the molar ratio of the maltohexaose to the beta-cyclodextrin being 5:1, and the labeling specific steps are as follows:
s1, weighing 9.9mg of maltohexaose and 11.34mg of beta-cyclodextrin, preparing into 1mmol/L aqueous solution respectively, mixing the maltohexaose and the beta-cyclodextrin solution according to the molar ratio of 5:1, and analyzing the obtained mixture solution by MALDI-TOF MS for later use.
S2, weighing 18.7mg GP reagent and dissolving in 1mL solvent (methanol: water: glacial acetic acid: 6:3:1) to prepare 0.1M GP reaction solution; 16.7mg of GT reagent was dissolved in 1mL of a solvent (methanol: water: glacial acetic acid: 6:3:1) to prepare a 0.1M GT reaction solution.
And (3) centrifuging, concentrating and drying the mixture samples in S3 and S1, adding 25uL of GP reaction liquid and GT reaction liquid respectively, reacting in a water bath at 70 ℃ for 1h, centrifuging, concentrating to remove the solvent, dissolving with 1mL of ultrapure water respectively, and using for MALDI-TOF MS detection.
The final samples of the experiments of example 2 and comparative example 2-1 were subjected to MALDI-TOF MS detection analysis, and the obtained mass spectra are shown in FIG. 3, in which FIG. 3A is a MALDI-TOF MS spectrum of an underivatized sugar chain mixture in which M/z 1013.75 and M/z 1029.53 are [ M + Na ] of maltohexaose, respectively]+Form [ M + K ]]+The ion signal peaks of the forms, M/z 1158.23 and M/z 1174.19 are [ M + Na ] of beta-cyclodextrin respectively]+Form [ M + K ]]+(ii) a formal ion signal peak, the mass spectrum signal intensity ratio between maltohexaose and beta-cyclodextrin is about 1:15, but the molar ratio of maltohexaose to beta-cyclodextrin is actually 5:1, because beta-cyclodextrin has better hydrophobicity than maltohexaose and is more easily ionized in a mass spectrometry ion source; FIG. 3B is a MALDI-TOF MS spectrum of a maltohexaose labeled GT reagent wherein M/z 1104.95 is [ M ] of a GT derivative of maltohexaose]+Form, and β -cyclodextrin is not derivatized; FIG. 3C is a MALDI-TOF MS spectrum of maltohexaose labeled with GP reagent, wherein M/z 1124.99 is [ M ] of GP derivative of maltohexaose]Form + and β -cyclodextrin is not derivatized; FIG. 3D is a MALDI-TOF MS spectrum of maltohexaose labeled with HMP reagent, wherein M/z 1138.57 is [ M ] of HMP derivative of maltohexaose]+Form, and β -cyclodextrin is not derivatized. The degree of improvement of sensitivity of three reagents, namely GP, GT and HMP, on the maltohexaose mass spectrum detection is compared by taking beta-cyclodextrin as an internal standard, the result shows that the improvement of the sensitivity of the reagent HMP on the reducing sugar chain mass spectrum detection is maximum, and the HMP labeling method has obvious advantages in the aspect of improving the sugar chain detection sensitivity.
Example 3
A general low-cost quaternary ammonium salt sugar chain isotope labeling reagent is used for investigating quantitative linear range and quantitative stability, a sugar chain standard substance is maltodextrin, and the method comprises the following specific steps:
s1, 1mg of maltodextrin was weighed out and dissolved in 0.5mL of ultrapure water to prepare a sugar chain standard solution.
S2, 24.5mg of HMP was dissolved in 1mL of a solvent (methanol: water: glacial acetic acid: 6:3:1) to prepare 0.1M d0-a HMP reaction solution; 31.5mg d are weighed out7HMP dissolved in 1mL of solutionFormulation (methanol: water: glacial acetic acid: 6:3:1) was formulated to 0.1M d7-HMP reaction solution.
S3, 1uL of sugar chain standard solution is taken by a micro-syringe and added into a 0.5mL centrifuge tube to prepare 2 parts in total, and then 25uLd parts are added respectively0HMP reaction solution and d7The HMP reaction solution was reacted in a water bath at 70 ℃ for 1 hour, and then centrifuged, concentrated and dried to remove the solvent, and finally dissolved in 1mL of ultrapure water.
S4, mixing the two solutions obtained in S3 by using a micro-sampling needle according to the volume ratio of 1:10, 1:7, 1:5, 1:3, 1:2, 1:1, 2:1, 3:1, 5:1, 7:1 and 10:1, and analyzing by MALDI-TOF MS detection.
The results of example 3 are shown in fig. 4, in which fig. 4A is a MALDI-TOF MS detection spectrum of a sample in which the d0-HMP derivative of maltodextrin and the d7-HMP derivative were mixed in four different ratios, and the results show that the ratio of the intensities of the corresponding light and heavy isotope ion signal peaks presented in the mass spectrum is consistent with the molar ratio of the corresponding light and heavy isotope derivatives; FIG. 4B is a linear relationship between the ratio of the signal peak intensities of the light and heavy isotopes of the maltodextrin fractions having polymerization degrees of 10, 12, 15, 17 and 18 and the molar ratio of the light and heavy isotope derivatives, and the results show that there is a linear relationship between the molar ratio of the light and heavy isotope-labeled maltodextrin oligosaccharides and the signal intensity ratio thereof in the mass spectrum, the linear range is 0.1-10, and the regression curve R2All of them were more than 0.98, indicating that the reagent of the present invention has higher reliability for the sugar chain relative to the quantitative analysis.
Example 4
Universal low-cost quaternary ammonium salt sugar chain isotope labeling reagent d0/d7HMP with the existing stable isotope reagent d0/d5And (3) comparing the mass spectrum detection signal resolution of the GP on the products marked by the reducing sugar chains with different molecular weight segments, wherein the selected sugar chain standard is maltodextrin. The procedure was as in example 3 except that the ratio of the light isotope reagent label to the heavy isotope label in maltodextrin was 1: 1.
Comparative example 4-1
S1, 1mg of maltodextrin was weighed and dissolved in 0.5mL of ultrapure water to prepare a sugar chain standard solution.
S2, 18.7mg of GP reagent was weighed out and dissolved in 1mL of solvent (methanol: water: glacial acetic acid: 6:3:1) to prepare 1mmol/Ld0-GP reaction solution; 23.7mg d are weighed out5The (co) -GP reagent was dissolved in 1mL of a solvent (methanol: water: glacial acetic acid ═ 6:3:1) to prepare 0.1M d5-GP reaction solution.
S3, adding 1uL of sugar chain standard solution into a 0.5mL centrifuge tube by using a micro-injection needle, preparing 2 parts simultaneously, and adding 25uL d0-GP solution and d5And (3) uniformly mixing the-GP solution, reacting in a water bath at 70 ℃ for 1h, and then centrifuging, concentrating and drying to remove the solvent.
S4, dissolving d of the obtained maltodextrin respectively with 1mL of ultrapure water0-GP marker and d5GP marker, then the same volume of sample is taken from the two solutions for mixing, and after thorough mixing, the mixture is analyzed by MALDI-TOF MS detection.
MALDI-TOF MS spectra of example 4 and comparative example 4-1 are shown in FIG. 5. FIG. 5A shows d of maltodextrin0-GP and d5MALDI-TOF MS spectrum of equal proportion mixture of GP markers, it can be seen that when the polymerization degree of oligosaccharide molecules in maltodextrin is increased to 15, the mass spectrum signal peaks of corresponding light and heavy isotope markers have partial overlap, relative quantitative accuracy is difficult to guarantee, and when the polymerization degree reaches 18, the mass spectrum signal peaks of corresponding light and heavy isotope markers have complete overlap, relative quantitative analysis cannot be carried out. FIG. 5B shows d of maltodextrin0-HMP and d7MALDI-TOF MS spectrum of equal proportion mixture of HMP marker, it can be seen that when the polymerization degree of oligosaccharide molecules in maltodextrin reaches 20, the mass spectrum signal peaks of corresponding light and heavy isotope markers can be still well distinguished, and it can be proved that d of the invention0-/d7The HMP reagent is suitable for the relative quantitative analysis of sugar chains with larger molecular weight, and the application range of the HMP reagent to the sugar chains is obviously larger than d0-/d5-a GP reagent.
Example 5
The method for evaluating the capability of a general low-cost quaternary ammonium salt sugar chain isotope labeling reagent on high-sensitivity mass spectrum detection of neutral N-sugar chains selects a glycoprotein standard substance as egg albumin, and comprises the following specific steps:
s1, 5mg of a sample of chicken albumin was weighed, dissolved in 450. mu.L of ultrapure water, 50. mu.L of a protein denaturing solution (50mg of SDS and 62mg of DTT in 1mL of ultrapure water) was added thereto, and the mixture was denatured by heating at 100 ℃ for 10 min. When the sample was cooled to room temperature, 50. mu.L of an enzymatic buffer (1.9g sodium phosphate in 10mL double distilled water, pH adjusted to 7.5 with phosphoric acid), 50. mu.L of P-40 (10%, v/v) and 1.0. mu.L of PNGase F enzyme were added and reacted at 37 ℃ for 24 h. After the reaction is finished, re-dissolving the sample in 1mL double distilled water after decompression and concentration, sequentially purifying by a C18 solid phase extraction column and a graphite carbon solid phase extraction column to obtain a reducing neutral N-sugar chain sample, detecting and analyzing by MALDI-TOF MS, centrifuging, concentrating and drying;
s2, adding 25uL of d in example 1 to the resulting chicken protein neutral N-sugar chain sample0Heating the HMP reaction solution in a water bath at 70 ℃ for 1h for reaction, centrifuging, concentrating to remove the solvent, dissolving with 0.5mL of ultrapure water, and using for MALDI-TOF MS detection analysis.
FIG. 6-A is a MALDI-TOF MS spectrum of underivatized egg white protein N-sugar chains, with 19 neutral N-sugar chains with different monosaccharide compositions detected in total, respectively: h3N2、H4N2、H3N3、H5N2、H4N3、H3N4、H6N2、H5N3、H4N4、H3N5、H7N2、H5N4、H4N5、H3N6、H5N5、H4N6、H3N7、H4N7、H3N8Wherein H represents a hexose and N represents N-acetamidohexose. Most of sugar chains are represented by [ M + Na ] in mass spectrum]+And [ M + K]+The two adducted ion forms are present, and the sugar chain with higher abundance also has [ M + H]+Signal peaks of the form in which H appears4N2Sum of potassium ion addition peak and H3N3The hydrogen ion addition peaks of (a) coincide at m/z 1114.08. FIG. 6-B is a MALDI-TOF MS spectrum of an HMP marker for an N-sugar chain of chicken protein, in which 23N-sugar chains were detected in total, and they were: 1058.33 (H)3N2)、1220.33(H4N2)、1261.33(H3N3)、1382.42(H5N2)、1423.55(H4N3)、1464.33(H3N4)、1544.33(H6N2)、1585.42(H5N3)、1627.42(H4N4)、1667.42(H3N5)、1706.55(H7N2)、1746.33(H6N3)、1788.50(H5N4)、1829.58(H3N6)、1870.42(H5N5)、1991.50(H4N6)、2032.64(H4N6)、2073.63(H3N7)、2153.86(H6N5)、2235.83(H4N7)、2277.12(H3N8)、2397.25(H5N7) And 2438.55 (H)4N8) Wherein the numerical value represents the m/z value of the sugar chain in the mass spectrum. Each sugar chain is represented by [ M ] in a mass spectrum]+The form presentation not only greatly reduces the types and the number of ion signals in the map, reduces the data analysis difficulty, effectively avoids the interference of impurity signals, but also greatly improves the detection sensitivity of sugar chains, can detect more low-abundance sugar chains, and fully proves that the reagent has good applicability and great advantage in neutral N-sugar chain analysis.
Example 6
The method for evaluating the capability of a universal low-cost quaternary ammonium salt sugar chain isotope labeling reagent HMP to quantitative analysis of sialylated N-sugar chains of a glycoprotein sample comprises the following specific steps of:
s1, the same procedure as in S1 of example 5 except that 5mg of chicken albumin was replaced with 5mg of fetal bovine serum albumin;
s2, dissolving the sialylated N-sugar chain of the fetal calf serum obtained in S1 in 25uL of isopropylamine reaction liquid (96mg of EDC.HCl, 67.5mg of HOBt and 191mg of isopropylamine hydrochloride in 1mL of DMSO), heating in a water bath at 60 ℃ for reaction for 2h, cooling to room temperature after the reaction is finished, adding 1.5mLACN to quench the reaction, collecting the precipitate after high-speed centrifugation at 13000r/min for 3min, dissolving the precipitate in 30uL of 4% methylamine water solution, adding 25uL of methylamine reaction liquid (65mg of PyBop, 33.75mg of methylamine hydrochloride and 76.5 uLN-methylmorpholine in 1mL of DMSO) after centrifugal concentration and drying, reacting in the water bath at 37 ℃ for 1.5h, adding 1.5mLACN to quench the reaction after the reaction is finished, centrifuging at 13000r/min at high speed for 3min, removing the supernatant, and collecting the precipitate;
s3, adding 25uL of the HMP reaction solution of example 1 to the obtained precipitate, reacting in a water bath at 70 ℃ for 1 hour, centrifuging, concentrating, drying, dissolving in 0.5mL of ultrapure water, and analyzing by MALDI-TOF MS.
FIG. 7 is a MALDI-TOF MS detection map of a derivative obtained by HMP labeling of an amidated modification product of a sialylated N-sugar chain of fetal bovine serum, 29 sialylated N-sugar chain isomers are detected in total, and compared with the literature, some N-glycolylneuraminic acid modified N-sugar chains with low abundance can also be detected, and more sugar chain structures are found, so that the reagent is proved to be suitable for mass spectrum high-sensitivity detection and analysis of the sialylated N-sugar chains subjected to amidation modification.
Example 7
Universal low-cost quaternary ammonium salt sugar chain isotope labeling reagent d0/d7-evaluation of the capability of quantitative analysis of fucosylated modified neutral N-sugar chains in a biological sample by HMP, wherein the selected biological sample is human semen total protein, and the specific steps are as follows:
s1, the same procedure as in S1 of example 5 except that 5mg of chicken albumin was replaced with 5mg of total protein of human semen;
s2, dividing the obtained human semen N-sugar chain into two equal parts, wherein 25uL d is added into one part0HMP reaction solution, another 25uL d7HMP reaction solution, heating in 70 deg.C water bath for 1 hr, mixing the obtained samples, and detecting with MALDI-TOF MS.
FIG. 8 shows d of N-sugar chain in human seminal fluid0-HMP and d7Of an equal proportion of a mixture of HMP derivativesMALDI-TOF MS detection spectrum, and 14 pairs of stable isotope reagent d are detected0/d7[ M ] of HMP-labeled sugar chain]+Form ion signal peak, high detection sensitivity, clear distinction between all pairs of isotope mass spectrum peaks, and proof of the invention0/d7The HMP reagent is suitable for mass spectrum high-sensitivity detection and quantification of high-fucosylated large molecular weight N-sugar chains in biological samples.
Example 8
Universal low-cost quaternary ammonium salt sugar chain isotope labeling reagent d0/d7The evaluation of the relative quantitative analysis capability of HMP on various N-sugar chains in different biological samples, wherein the selected biological samples are total protein of pig serum and total protein of chicken serum, and the specific steps are as follows:
s1, the same as the operation S1 in example 5, except that 5mg of chicken albumin is replaced with 5mg of total porcine serum protein;
s2, the same as the operation S1 in example 5, except that 5mg of chicken albumin is replaced with 5mg of chicken serum total protein;
s3, the same as the procedure of S2 in example 6, except that the sugar chain sample used was N-sugar chains released from porcine serum;
s4, the same as the procedure of S2 in example 6, except that the sugar chain sample used was N-sugar chains released from chicken serum;
s5, adding 25uL d to the sugar chain amidated and modified in the step S37HMP, heating in a water bath at 70 ℃ for 1h, and detecting and analyzing the obtained sample by MALDI-TOF MS; for the sugar chain amidated and modified in the S4 step, 25uL d was added0HMP, heating in a water bath at 70 ℃ for 1h, and detecting and analyzing the obtained sample by MALDI-TOF MS; the two samples obtained were mixed in equal total protein sample equivalents and analyzed by MALDI-TOF MS detection.
D of N-sugar chain of total protein in chicken serum0-HMP derivatives, d of N-sugar chains of total protein of porcine serum7MALDI-TOF MS spectra of the HMP derivative and an equal ratio mixture of the two are shown in FIG. 9. FIG. 9A shows amidation modification of N-sugar chains released from total chicken serum proteinsAnd d0MALDI-TOF MS spectrum after HMP labelling, with a total detection of 33N-sugar chains (including isomers), respectively: 1261.04 (H)3N3)、1382.66(H5N2)、1424.61(H4N3)、1544.89(H6N2)、1569.32(H4N3F)、1585.59(H5N3)、1626.89(H4N4)、1706.55(H6N2)、1731.19(H5N3F)、1747.23(H6N3)、1772.25(H4N4F)、1788.01(H5N4)、1830.23(H4N5)、1870.25(H3N6)、1901.89(H4N3FAc-α2,6)、1934.33(H5N4F)、1991.88(H5N5)、2031.86(H9N2)、2079.55(H6N3Ac-α2,6)、2092.18(H5N4Ac-α2,3)、2104.33(H4N4FAc-α2,6)、2120.05(H5N4Ac-α2,6)、2136.55(H5N4Gc-α2,6)、2192.35(H10N2)、2238.13(H5N4FAc-α2,3)、2266.35(H5N4FAc-α2,6)、2424.25(H5N4Ac2-α2,3+α2,6)、2452.78(H5N4Ac2-α2,6+α2,6)、2468.33(H5N4AcGc-α2,6)、2484.66(H5N4Gc2-α2,6+α2,6)、2789.33(H6N5Ac2-α2,3+α2,6)、2817.55(H6N5Ac2-α2,6+α2,6)、2849.45(H6N5Gc2- α 2,6+ α 2,6), wherein F represents fucose, Ac represents N-acetylneuraminic acid, Gc represents N-glycolylneuraminic acid, the latter short line describing the attachment of sialic acid on the sugar chain; FIG. 9B shows amidation modification reaction of N-sugar chain released from total protein of pig serum and d7MALDI-TOF MS spectrum after HMP labeling, with total detection of 28N-sugar chains (including isomers), 1267.56 (H) respectively3N3)，1388.56(H5N2)，1429.33(H4N3)，1470.32(H3N4)，1551.39(H6N2)，1592.07(H5N3)，1617.19(H3N4F)，1633.36(H4N4)，1794.54(H5N4)，1941.48(H5N4F)，2248.69(H5N4FAc-α2,3)，2273.97(H5N4FAc-α2,6)，1227.56(H4N2)，1413.24(H3N3F)，1575.05(H4N3F)，1551.39(H6N2)，2037.04(H9N2)，1907.88(H4N3FGc-α2,3)，2085.76(H4N4FAc-α2,3)，2127.68(H5N4Ac-α2,6)，2248.69(H5N4FAc-α2,3)，2273.97(H5N4FAc-α2,6)，2289.99(H5N4FGc-α2,6)，2459.70(H5N4Ac2-α2,6+α2,6)，2578.43(H5N4FAc2-α2,3+α2,6)，2606.76(H5N4FAc2-α2,6+α2,6)，2621.08(H5N4FAcGc- α 2, 6); FIG. 9C shows amidation-modified and d0/d N-sugar chains released from equal amounts of total porcine and chicken serum proteins7MALDI-TOF MS spectrum of the product obtained by mixing in equal proportion after HMP labeling can directly reflect the content ratio of each sugar chain in pig serum and chicken serum. FIG. 9A and FIG. 9B show that 30N-sugar chains are present in both pig serum and chicken serum, and the ion signal peaks of the d0/d7-HMP markers of these sugar chains are also detected in FIG. 9C.
The results of relative quantitative analysis of various N-sugar chains in pig serum and chicken serum are shown in Table 1. As can be seen from Table 1, by analyzing the sugar chain d0/d7After the data of the ratio of the isotope to the peak signal intensity of the HMP derivative, it was found that the content of 15N-sugar chains in chicken serum was lower than that in pig serum, and the content of 14N-sugar chains was higher than that in pig serum. Wherein, chicken serum 2120.55/2127.65 (H)5N4Ac-α2,6)、1991.25/1998.78(H5N5) And 2452.33/2459.45 (H)5N4Ac2The content of- alpha 2,6+ alpha 2,6) is far higher than that of pig serum and reaches 10.5 times, 6.0 times and 5.54 times respectively; while in chicken serum 1934.25/1941.33 (H)5N4F)、1772.45/1779.33(H4N4F)、1901.45/1908.25(H4N3FAc-. alpha.2, 3) and 1464.85/1471.25 (H)3N4) The content of the (D) is far lower than that of pig serum, and is only 0.15, 0.18, 0.27 and 0.28 times of the content of the (D) in pig serum.
TABLE 1 results of quantitative comparative analysis of N-sugar chains in pig serum and chicken serum
Attached notes: black squares in the table indicate N-acetylglucosaminyl sugars; dark gray circles indicate mannose; light gray circle represents galactose; dark triangles indicate fucose; dark diamonds represent N-acetylneuraminic acid; the light diamonds represent N-glycolylneuraminic acid.
These results fully demonstrate that the HMP reagent of the invention is suitable for mass spectrum high-sensitivity relative quantitative analysis of biological reducing sugar chains with different molecular weight segments, and can be used for comparing content differences of various N-sugar chains in different biological samples.
The stable isotope reagent deuterium 7-4-methyl-1- (2-hydrazino-2-oxoethyl) -pyridinium bromide and 4-methyl-1- (2-hydrazino-2-oxoethyl) -pyridinium bromide provided by the invention are quaternary ammonium compounds with hydrazide groups, can be marked at the reducing end of a biological sugar chain under mild weak acid conditions,the positive charges of the compounds can simplify the peak form and greatly improve the sensitivity of mass spectrum detection in the mass spectrum analysis of the reducing sugar chains; deuterium 7-4-methyl-1- (2-hydrazino-2-oxoethyl) -pyridinium and 4-methyl-1- (2-hydrazino-2-oxoethyl) -pyridinium have a relative molecular mass difference of 7Da and are matched to form a mass difference of 7Da, so that the mass difference has good resolution in mass spectrum relative quantitative analysis of the bioreductive sugar chains in molecular weight ranges above 2000Da and is also suitable for molecular weight ranges below 2000 Da; the synthetic raw material deuterium 7-4-methylpyridine of the stable isotope reagent deuterium 7-4-methyl-1- (2-hydrazino-2-oxoethyl) -pyridinium provided by the invention is a commercial reagent, compared with d7Starting materials d for the synthesis of HIQB7-isoquinoline, d7The price of the-4 methylpyridine is cheaper, the development cost is lower, and the wide application can be realized.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (10)
1. A general low-cost quaternary ammonium salt sugar chain isotope labeling reagent is characterized by comprising a heavy isotope labeling reagent and a corresponding light isotope labeling reagent, wherein the heavy isotope labeling reagent has a structural formula shown in a formula (I):
the structural formula of the labeling reagent in the form of the light isotope is shown as a formula (II):
2. the method for synthesizing a general-purpose low-cost quaternary ammonium salt-based sugar chain isotope labeling reagent according to claim 1, comprising the steps of:
s1, taking deuterated 4-methylpyridine or 4-methylpyridine and ethyl bromoacetate as raw materials, and generating a mixture containing a compound shown in a formula (III) through a nucleophilic substitution reaction between amine and alkyl halide at 65-70 ℃;
s2, adding hydrazine hydrate into the mixture S1 in an ice bath environment, and preparing a compound of a formula (I) or a compound of a formula (II) through nucleophilic substitution reaction between ester and amine, wherein deuterated 7-4-methyl-1- (2-hydrazino-2-oxoethyl) -pyridinium bromide or 4-methyl-1- (2-hydrazino-2-oxoethyl) -pyridinium bromide;
the synthetic route is as follows:
3. the method for synthesizing a universal low-cost quaternary ammonium salt-based sugar chain isotope labeling reagent according to claim 2, wherein the solvent in S1 is methanol or ethanol.
4. The method for synthesizing a general-purpose low-cost quaternary ammonium salt-based sugar chain isotope labeling reagent according to claim 2, wherein in S1, ethyl bromoacetate: the molar ratio of the deuterated 4-methylpyridine or the 4-methylpyridine is 1.1-1.5: 1.
5. The method for synthesizing a general-purpose low-cost quaternary ammonium salt-based sugar chain isotope labeling reagent according to claim 2, wherein the reaction time in S1 is 12 hours.
6. The method for synthesizing a universal low-cost quaternary ammonium salt-based sugar chain isotope labeling reagent according to claim 2, wherein hydrazine hydrate is dropwise added to the S1 mixture under stirring in S2, and the reaction is continued for 2 hours in an ice bath environment after the dropwise addition is completed within 30 minutes.
7. The method for synthesizing a general-purpose low-cost quaternary ammonium salt-based sugar chain isotope labeling reagent according to claim 2, wherein in S2, hydrazine hydrate: the molar ratio of ethyl bromoacetate is 1.1-1.5: 1.
8. The method for synthesizing a universal low-cost quaternary ammonium salt sugar chain isotope labeling reagent according to claim 2, wherein in S2, after the reaction is finished, the reaction product is concentrated, then precipitated with pre-cooled absolute ethanol, filtered, washed and dried to obtain the product.
9. A relative quantitative analysis method for a biological reducing sugar chain mass spectrum is characterized by comprising the following steps:
carrying out hydrazone reaction on two reducing sugar chain samples derived from an equivalent biological sample, and the compound of formula (II) and the compound of formula (I) respectively under the condition that the pH value is 2-5 to prepare a first mixture and a second mixture, mixing the first mixture and the second mixture, concentrating to prepare an aqueous solution, and carrying out detection and analysis by mass spectrometry.
10. The method of claim 9, wherein the mass spectrometry is electrospray ionization mass spectrometry or matrix-assisted laser desorption ionization-time of flight mass spectrometry.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011553376.7A CN112778194B (en) | 2020-12-24 | 2020-12-24 | Universal low-cost quaternary ammonium salt sugar chain isotope labeling reagent and synthetic method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011553376.7A CN112778194B (en) | 2020-12-24 | 2020-12-24 | Universal low-cost quaternary ammonium salt sugar chain isotope labeling reagent and synthetic method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112778194A true CN112778194A (en) | 2021-05-11 |
| CN112778194B CN112778194B (en) | 2022-08-05 |
Family
ID=75752182
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011553376.7A Active CN112778194B (en) | 2020-12-24 | 2020-12-24 | Universal low-cost quaternary ammonium salt sugar chain isotope labeling reagent and synthetic method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112778194B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115508400A (en) * | 2022-11-23 | 2022-12-23 | 中国石油大学(华东) | Method for determining content of benzyl quinoline quaternary ammonium salt dimer |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101759633A (en) * | 2010-01-15 | 2010-06-30 | 延边大学 | Reagents for analyzing N-linked sugar chain |
| CN102175750A (en) * | 2011-02-25 | 2011-09-07 | 中国科学院上海有机化学研究所 | Method for analyzing biological samples by using matrix assisted laser desorption ionization-Fourier transform ion cyclotron resonance mass spectra |
-
2020
- 2020-12-24 CN CN202011553376.7A patent/CN112778194B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101759633A (en) * | 2010-01-15 | 2010-06-30 | 延边大学 | Reagents for analyzing N-linked sugar chain |
| CN102175750A (en) * | 2011-02-25 | 2011-09-07 | 中国科学院上海有机化学研究所 | Method for analyzing biological samples by using matrix assisted laser desorption ionization-Fourier transform ion cyclotron resonance mass spectra |
Non-Patent Citations (5)
| Title |
|---|
| MIKI KAMEMURA等: "Sample-multiplexing by derivatization using multiple analogous reagents for enhancing throughput in LC/ESI-MS/MS assay of steroids: Plasma 17α-hydroxyprogesterone as an example", 《JOURNAL OF CHROMATOGRAPHY B》 * |
| 何金霞等: "植物N-糖链检测技术研究进展", 《生物技术进展》 * |
| 吴智宇: "基于稳定同位素标记吉拉德试剂P的还原性寡糖相对定量分析法", 《中国优秀硕士论文全文数据库》 * |
| 邓春晖等: "《磁性为纳米材料在蛋白质组学中的应用》", 31 December 2017, 复旦大学出版社 * |
| 黄江铭等: "基于质谱的糖蛋白/糖链定量技术研究进展", 《分析科学学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115508400A (en) * | 2022-11-23 | 2022-12-23 | 中国石油大学(华东) | Method for determining content of benzyl quinoline quaternary ammonium salt dimer |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112778194B (en) | 2022-08-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhang et al. | Sensitive and robust MALDI-TOF-MS glycomics analysis enabled by Girard's reagent T on-target derivatization (GTOD) of reducing glycans | |
| JP5396277B2 (en) | Automatic sugar chain pretreatment equipment | |
| Kailemia et al. | Oligosaccharide analysis by mass spectrometry: a review of recent developments | |
| Harvey | Structural determination of N‐linked glycans by matrix‐assisted laser desorption/ionization and electrospray ionization mass spectrometry | |
| JP4583168B2 (en) | Method for quantitative proteome analysis of glycoproteins | |
| Zhang et al. | Relative quantitation of glycans using stable isotopic labels 1-(d0/d5) phenyl-3-methyl-5-pyrazolone by mass spectrometry | |
| Liu et al. | Methylamidation for sialoglycomics by MALDI-MS: a facile derivatization strategy for both α2, 3-and α2, 6-linked sialic acids | |
| EP0708837B1 (en) | Fluorescent polymer labeled conjugates and intermediates | |
| Harvey | Negative ion mass spectrometry for the analysis of N‐linked glycans | |
| Nishimura | Toward automated glycan analysis | |
| Nishikaze et al. | Structural analysis of N-glycans by the glycan-labeling method using 3-aminoquinoline-based liquid matrix in negative-ion MALDI-MS | |
| CN109541115B (en) | High-resolution sequential separation and accurate quantitative analysis method for sialylated sugar chain isomers | |
| CN110487888B (en) | Application of DHB/DHBH in combination type matrix in detection of reducing sugar in MALDI mass spectrometry | |
| JP4328625B2 (en) | Labeling reagents and their use | |
| Walker et al. | Systematic comparison of reverse phase and hydrophilic interaction liquid chromatography platforms for the analysis of N-linked glycans | |
| EP2406634A2 (en) | Deuterium isobaric tag reagents for quantitative analysis | |
| EP1617223A2 (en) | Serial derivatization of peptides for "de Novo" sequencing using tandem mass spectrometry | |
| US20090250604A1 (en) | Analysis Method for Biological Sample and Screening Method for Disease Marker | |
| CN112778194B (en) | Universal low-cost quaternary ammonium salt sugar chain isotope labeling reagent and synthetic method thereof | |
| CN102944604A (en) | Use of piperazinopyrimidine isotope labeling reagent | |
| CN110028539B (en) | Isotope labeled bionic sugar or sugar group, preparation method and application thereof | |
| TWI547486B (en) | Compounds and methods for analysis and synthesis of saccharide compounds | |
| Ma et al. | Carboxyl-based CPMP tag for ultrasensitive analysis of disaccharides by negative tandem mass spectrometry | |
| Wang et al. | Glycan reductive amino acid coded affinity tagging (GRACAT) for highly specific analysis of N-glycome by mass spectrometry | |
| EP1916526A1 (en) | Method for diagnostic and therapeutic target discovery by combining isotopic and isobaric labels |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |