CN105131035B - Amino group compound and sugar chain labeled mass spectrum derivatization reagent with positive charges - Google Patents
Amino group compound and sugar chain labeled mass spectrum derivatization reagent with positive charges Download PDFInfo
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Abstract
The invention discloses an amino group compound and a N-linked sugar chain labeled mass spectrum derivatization reagent with positive charges, and specifically relates to an MPST reagent. The reagent has a triphenyl phosphine chemical structure with positive charges, can be used to label amino group compounds and N-linked sugar chain chemically, and is capable of cooperating with a liquid chromatography-mass spectrum (LC-MS) technology to establish a high-sensitive and high-selective analysis method of amino group compounds and N-linked sugar chain. Because the reagent carries positive charges, at the same time, specific fragment ions with a mass/charge ratio (m/z) of 120 can be obtained in MS/MS, the detection sensitivity of mass spectrum can be prominently improved, and the structure can be easily determined under the mode of electrospray ionized positive ions (ESI+). A novel high-sensitive mass spectrum reagent (fig. 3) with positive charges is provided for the research on biological metabonomics and biological function of trace sugar chain and screening of biological markers of various diseases.
Description
Technical field
The present invention relates to the metabolite analysis containing amido functional group of field of bioanalysis and diversity sugar group field, especially
It is containing amino-functional compounds and N- connection sugar chain analysis high sensitivity mass spectrum derivatization reagents.
Background technology
Metabolism group (Metabonomics) is the monolithic of the biological endogenous property metabolite of quantitative description and its internally
The rule sexology that external environment change is responded.Concentration of the metabolism group by measurement cell, tissue and organism metabolism product
Change is reflecting gene, the protein and metabolic activity direct relation of itself.Due to the change of metabolism group be body to gene,
The effects such as disease, environment and medicine are finally reflected, its endogenous metabolites be a series of the crucial of body vital movements or
End reaction.Therefore, metabolism group can help people to be best understood from various complicated interactions and its sheet in organism
Matter.In recent years, metabolism group is in the research side such as medical diagnosis on disease and animal model, drug metabolism, the safety evaluatio of Chinese medicine ingredients
Face achieves new breakthrough and progress.But its analysis method remains one of bottleneck that metabolism group is developed rapidly.
The metabolism group research method of early stage mainly adopts nuclear magnetic resonance, NMR (NMR) technology, gas phase-mass spectrum（GC-MS）Technology
And capillary electrophoresis-mass spectrometry（CE-MS）Technology.The major advantage of NMR is that condition determination is gentle, does not change the structure and reason of sample
Change property, can also carry out dynamic measurement, but have detection sensitivity low, the limitation such as narrow dynamic range.Under comparing, LC-MS
Multiple techniques, using remarkable separating power and the high duplication of liquid technology, the multi-channel testing function of mass-spectrometric technique, particularly
The characteristics of high resolution mass spec is by its universality, high sensitivity and wider dynamic detection range, specificity, it has also become metabolism
Group learns the mainstream technology of research.Can be applicable to fixedness compound, polar compound, heat-labile compound and macromolecule
The analysis of compound.But also have to some metabolite because the low detection sensitivity of Ionization Efficiency does not reach requirement, especially to complexity
Metabolite in biological sample is difficult to reach the purpose for improving selectivity and high-sensitivity detection.
In addition, sugar group is learned（Glycomics）It is expression, regulation and control and of physiological function for studying all sugar chains on glycoprotein
Section.The research that sugar group is learned is not only the continuity of genomics and proteomics, and be thoroughly illustrate gene function must be by
Road, and the Article 3 approach for cracking life-information.Sugar group learn focus of attention be glycoprotein, in order to preferably with protein
Group learns associated, so object of study to be locked as the sugar chain of glycoprotein.Link glycoprotein candy chain mainly has N- connections and O- to connect
Connect, the agedoite with protein（Asn）The amino side chain covalent bond of residue is connected to N- sugar chains, with the serine of protein or
Threonine（Ser/Thr）O- sugar chains are connected on hydroxyl oxygen.And the biological sugar chain of major part in vivo is N- sugar chains, and mainly position
In surface of cell membrane and memebrane protein, its cancer to cell and transfer, and hemopoietic function play an important role.Therefore, at this
Will be with N- sugar chains as analyzing object in invention research.
Up to the present, nuclear magnetic resonance, NMR（NMR）Technology has become the effective tool of sugar chain stereochemical structure analysis.Mainly
Advantage is that condition determination is gentle, neither changes the structure and physicochemical property of sample, can determine that the configuration of sugar again, link position, point
Prop up and microcosmic multiformity.But the signal peak overlapping that NMR determines sugar is serious, and it is more difficult to parse, and sensitivity is low, and needs milligram level sample
Product, this to most saccharide complexes in the analysis of micro sugar chain be extremely difficult to.In comparison, modern efficient liquid chromatograph-
Mass spectrum（LC-MS）Remarkable separating power and high duplication of the multiple techniques using liquid technology, the multi-channel testing of mass-spectrometric technique
Function, particularly high resolution mass spec rely on its universality, high sensitivity and wider dynamic detection range, the spy of specificity
Point, it has also become the mainstream technology of the qualitative and quantitative analysis of complicated micro sugar chain in biological sample.But also there are some biological specimens
It is non-renewable, and there is a problem of differentiating sugar chain stereochemical structure.The traditional method of analyzing glucoprotein sugar chain usually will
Sugar chain cuts away（Hydrazinolysis method and enzyme process）And be analyzed after isolating and purifying.Because sugar chain does not have chromophoric group in itself, and its
Ionizing is difficult on mass spectrograph, in order to more effectively detect sugar chain with during Structural Identification isolating and purifying, typically
The method for carrying out column front derivation.The method mainly makes ultraviolet on sugar chain band or fluorophor, improves the sensitivity of detection, together
When can make hydrophobic group on sugar chain band again, reduce the polarity of sugar chain, sugar chain is retained on reversed phase chromatographic column, beneficial to sugar
The separation of chain.The reagent for performing the derivatization labelling at present to sugar chain is more, mainly has 1-phenyl-3-methyl-5-pyrazolones ketone
(PMP), PA (2-AP), amino benzoic Acid esters derivative reagent (ABEE), phenyl aminess derivative reagent (2-AB).
Although can be used for detecting on fluorescence or MS after HPLC is separated after being marked sugar chain using these reagents, can with
Selectivity and the sensitivity of sugar chain analysis, but the more difficult control of chemical reaction condition of these reagents can be improved to a certain extent,
Different sugar chain structures need to grope different derivatising conditions, are not only susceptible to deglycosylation phenomenon, and need multistep journey
Its formality of sequence is loaded down with trivial details time-consuming.Wherein reductive amination method becomes one of conventional derivatization method as product is stable, but the party
Method is directly marked to the reducing end of sugar chain, and the recovery of raw sugar chain is relatively difficult, it may also happen that also during derivatization
There is the situation for being difficult to judge, and the sugar after deriving in former end isomerization, β-elimination reaction, sialic acid when losing and causing detection
Chain can form reducing end open loop structure, cause the part biological information loss and some activity to sugar chain of sugar chain to cause shadow
Ring.
In recent years, Kamoda (4 (2005) 146-152. of J. Proteome Res.) etc. utilizes N- glycosidase F
After (PNGase F) enzymolysis obtains complete N- sugar chains and peptide fragment, the reaction such as sugar chain reducing end and ammonium salt is made to generate intermediate product sugar
Ammonium (Glycosylamine), sugared ammonium are reacted with fluorescence derivatization again, develop N- sugar chain analysis methods.This analysis side
Although method keeps the original structure of sugar chain during sugar chain derivatization, the advantage of reducing end open loop will not be formed, because using
Fluorescence derivatization, does not reach detection in LC-MS sometimes and requires, and sugar chain structure parsing is difficult, is extremely difficult to the same time-division
Analyse the purpose of various sugar chains.
The content of the invention
The invention aims to overcome existing amido functional group metabolite and N- sugar chains in LC-MS detection sensitivities
It is not enough, there is provided one kind is per se with positive charge, and can obtain mass-to-charge ratio in MS/MS（m/z）For 120 particular fragment ion,
Can be in electro-spray ionization cation（ESI+）Under pattern, mass spectrographic detection sensitivity can be significantly improved, be easy to Structure Deduction
The positively charged mass spectrum derivatization reagent of amino-functional compounds and sugar chain labelling.
Technical scheme provided by the present invention is：
A kind of amino-functional compounds and the positively charged mass spectrum derivatization reagent of sugar chain labelling, with triphenylphosphine
(Triphenylphosphine, TPP) and 2- [4- (2-bromomethylphenyl)]-propanoic acid [2- [4- (Bromomethyl)
phenyl]-
Propionic acid (2-4-BMPPA)], N-hydroxy-succinamide (N-hydroxysuccinimide) is
Starting material, be synthesized [4- (methyl) phenyl-2-propionyl-N-succinimido]-
Triphenylphosphonium bromide (MPST), the MPST reagents have following structural formula：
。
The present invention, it is contemplated that carry out chemical derivatization method exploitation high sensitivity, high selectivity to amido functional group metabolite
LC-MS analysis methods；For N- sugar chains in glycoprotein, complete N- is obtained first with N- glycosidase F (PNGase F) enzymolysis
After sugar chain and peptide fragment, the reaction such as sugar chain reducing end and ammonium salt is made to generate intermediate product sugar ammonium, sugared ammonium is spread out with the new mass spectrum for synthesizing again
Biochemical reagents react, exploitation high sensitivity, high selectivity N- sugar chain analysis method.It is that biological metabolism group and micro sugar chain are biological
The further investigation of function and the screening of various disease biomarkers are provided effectively, reliably analyzes detection meanss.
Currently invention addresses positively charged, easy protonation in electron spray positive ionization mode (ESI+) in, be conducive to choosing
Selecting triphenylphosphine (Triphenylphospine) chemical constitution that reaction monitoring (SRM) or multiple-reaction monitoring (MRM) analyze is
Parent, develop with high sensitivity, high selectivity targeting amido functional group and meanwhile can labelling sugar chain positively charged mass spectrum
Derivatization reagent.The exploitation of positively charged mass spectrum derivatization reagent, by the organism to developing and setting up highly sensitive, high selectivity
Interior micro sugar chain analysis method and the method for metabolite analysis containing amido functional group play bottleneck effect, real effectively to exploring screening
Sugar chain biomarker and metabolism biological marker, it is significant.
The present invention relates to amino-functional compounds and N- connection sugar chain analyrical reagents, refer specifically to MPST reagents.The examination
Agent contains positively charged triphenylphosphine structure, sugar chain and ammonia in the glycoprotein that can be digested to N- glycosidase F (PNGase F)
Base functional group metabolite carries out chemical derivatization reaction；Using application of gas chromatorgraphy/mass technology, the amino official for setting up highly sensitive, high selectivity
Metabolite and N- link sugar chain simultaneous analysis methods can be rolled into a ball.Amido functional group metabolite and sugar chain derivative can be improved in mass spectrum
Detection sensitivity on detector, and it is easy to Structure Deduction.For the various disease lifes that biological metabolism group and diversity sugar group are learned
The screening of thing mark provides effective, reliable reagents for mass spectrogram.
Below in conjunction with the accompanying drawings the embodiment of the present invention is further described.
Description of the drawings
Mass chromatograms of the Fig. 1 for the LC-MS of embodiment MPST reagent.
Mass spectruies of the Fig. 2 for the LC-MS of embodiment MPST reagent.
Fig. 3 is embodiment MPST reagent and amino-functional compounds reaction structure formula.
Fig. 4 is embodiment MPST reagent and sugared ammonium（SGA）Reaction structure formula.
Specific embodiment
Embodiment
The synthesis of MPST:
Weigh 85.0 mg of MPT（0.2 mmol）It is dissolved in 6.0 mL acetonitrile solvents, adds DCC（N, N '-dicyclohexyl
Carbodiimide）41.2 mg（0.2 mmol), it is eventually adding 23.0 mg of N- hydroxysuccinimides（0.2 mmol）, room temperature stirs
Mix reaction 16 hours.Reactant liquor sucking filtration is removed into white powder, after being spin-dried for solvent, 127.4 mg products is obtained.Using washing
De- agent methanol:Dichloromethane=1:25 carry out column chromatography purification.ESI-MS spectral datas（m/z）：425.17 [M]+; 1H-
NMR： δ 1.56-1.59 (d, 3H,J =9 Hz), 2.84 (s, 4H), 3.93-4.00 (dd, H, J =12.5
Hz), 5.31-5.48 (d, 2H, J=51 Hz), 7.07-7.28 (m, 4H), 7.62-7.80 (m,15H).Fusing point is
128 oC, rate are about 94.9%：
。
It will be seen that the MPST reagent molecules of synthesis from the mass chromatogram of the LC-MS of Fig. 1, Fig. 2 and mass spectrum
Amount is consistent with mass-to-charge ratio m/z=522.18 for detecting.May determine that the compound strictly MPST reagents of synthesis.The concrete tables of Fig. 3
Show the MPST reagents of synthesis with the reactive site of amino-functional compounds and reaction structure formula.Fig. 4 specifically represents N- glycosidase F
(PNGase F) enzymolysis sialoglycopeptide Sialylglycopeptide (SGP) become saccharogenesis ammonium structure process, and with synthesis
MPST reagents perform the derivatization the reaction structure formula of reaction.
Reagent in the present invention contains positively charged triphenylphosphine structure, as this kind of reagents for mass spectrogram are per se with positive electricity
Lotus, can be in the electro-spray ionization cation of Mass Spectrometer Method（ESI+）Under pattern, can significantly improve determinand Ionization Efficiency,
And mass-to-charge ratio can be obtained in MS/MS（m/z）For 120 particular fragment ion, can react in Selective reaction monitoring (SRM) or more
Detection sensitivity is significantly improved in monitoring (MRM) pattern, and in precursor scans（precursor ion scan）Can profit
With the specific daughter ion of sugar chain of structure containing N-acetyl-glucosamine that m/z is 627, carry out while detect parent ion, carrying out Structure Deduction.
Serine with amido functional group, Phenylalanine, L-Valine, monosaccharide, D-glucose amine (D-Glucamine), sialoglycopeptide
Detection sensitivity after the derivatizations such as Sialylglycopeptide (SGP) is 0.3 amol-30 fmol.Apparently higher than now
Some fluorescence derivatizations and reagents for mass spectrogram.Using chromatography-mass spectroscopy（LC-MS）Multiple techniques, sets up amido functional group generation
Thank thing and N- connection the highly sensitive of sugar chain, the analysis method of high selectivity.It is that biological metabolism group and diversity sugar organize each
The screening for planting disease biomarkers provides the reagents for mass spectrogram of effective, reliable highly sensitive, high selectivity.
Claims (1)
1. the positively charged mass spectrum derivatization reagent of a kind of sugar chain labelling, with triphenylphosphine (Triphenylphosphine, TPP)
With 2- [4- (2-bromomethylphenyl)]-propanoic acid [2- [4- (Bromomethyl) phenyl]-
Propionic acid (2-4-BMPPA)], N-hydroxy-succinamide (N-hydroxysuccinimide) is starting
Thing, is synthesized [4- (methyl) phenyl-2-propionyl-N-succinimido]-triphenylphosphonium
Bromide (MPST), the MPST reagents have following structural formula：
。
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| CN106770838B (en) * | 2017-02-15 | 2019-05-14 | 延边大学 | Amido functional group chipal compounds split label band fluorescence derivatization |
| CN106905472B (en) * | 2017-04-18 | 2019-01-29 | 北京蛋白质组研究中心 | A kind of functionalization responsive to temperature type polymer and the preparation method and application thereof |
| CN108318614A (en) * | 2018-01-12 | 2018-07-24 | 中国科学院昆明植物研究所 | A kind of isotopic dilution LC-MS methods measuring inside/outside source property blood sugar concentration |
| CN111208254B (en) * | 2019-12-31 | 2023-09-22 | 延边大学 | Sugar chain relative quantitative isotope labeling mass spectrum derivatization reagent |
| CN115667934A (en) * | 2020-05-20 | 2023-01-31 | 豪夫迈·罗氏有限公司 | Reagent for mass spectrometry |
| CN111896666A (en) * | 2020-08-12 | 2020-11-06 | 黄河三角洲京博化工研究院有限公司 | Detection method of triphenylphosphine sodium tri-sulfonate |
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| CN101759633A (en) * | 2010-01-15 | 2010-06-30 | 延边大学 | Reagents for analyzing N-linked sugar chain |
| CN104181258A (en) * | 2013-05-24 | 2014-12-03 | 北京蛋白质组研究中心 | Glycoprotein N-carbohydrate chain one-step enrichment-derivation processing method based on graphene and MALDI-TOF-MS analysis method |
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