CN105131035A - 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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- CN105131035A CN105131035A CN201510470738.9A CN201510470738A CN105131035A CN 105131035 A CN105131035 A CN 105131035A CN 201510470738 A CN201510470738 A CN 201510470738A CN 105131035 A CN105131035 A CN 105131035A
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- sugar chain
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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
What the present invention relates to field of bioanalysis organizes field containing amido functional group metabolite analysis and otherness sugar, particularly connects sugar chain analysis highly sensitive mass spectrum derivatization reagent containing amino-functional compounds and N-.
Background technology
Metabolism group (Metabonomics) is the whole body shape Condition of quantitative description biological endogenous property metabolic substd and internal and external environment is changed to the regular science making response.Metabolism group reflects the direct relation of gene, protein and metabolic activity itself by the change in concentration measuring cell, tissue and organism metabolism product.Change due to metabolism group is the final reflection that body acts on gene, disease, environment and medicine etc., and its endogenous metabolites is key or the end reaction of a series of vital movement of body.Therefore, metabolism group can help people to understand interaction and the essence thereof of various complexity in organism better.In recent years, metabolism group achieves new breakthrough and progress in the research such as safety evaluation of medical diagnosis on disease and animal model, drug metabolism, traditional Chinese medicine ingredients.But its analytical procedure remains one of bottleneck that metabolism group develops rapidly.
Early stage metabolism group research method mainly adopts nucleus magnetic resonance (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 structure and the physico-chemical property of sample, also can carry out dynamic measurement, but have detection sensitivity low, the limitation such as narrow dynamic range.Under comparing, LC-MS coupling technique, utilize remarkable separating power and the high duplication of liquid technology, the multi-channel testing function of mass-spectrometric technique, particularly high resolution mass spec relies on its universality, highly sensitive and wider dynamic detection range, specific feature, has become the mainstream technology of metabolism group research.Can be applicable to the analysis of non-volatility compound, polar compound, heat-labile compound and macromolecule compound.But also have to some metabolites because the low detection sensitivity of Ionization Efficiency does not reach requirement, be especially difficult to reach the object improving selectivity and high-sensitivity detection to the meta-bolites in complex biological sample.
In addition, sugar group learns the subject that (Glycomics) is the expression of all sugar chains on research glycoprotein, regulation and control and physiological function.The continuity of genomics and proteomics is not only in the research that sugar group is learned, and is the only way of thoroughly illustrating gene function, is also the Article 3 approach cracking life-information.It is glycoprotein that sugar group learns the focus paid close attention to, in order to be associated with proteomics better, so research object to be locked as the sugar chain of glycoprotein.Link glycoprotein candy chain mainly contains N-and connects and be connected with O-, is connected to N-sugar chain, is connected to O-sugar chain with on the Serine of protein or Threonine (Ser/Thr) hydroxyl oxygen with the amino side-chain covalent linkage of l-asparagine (Asn) residue of protein.And most of sugar chain is N-sugar chain in organism, and be mainly positioned surface of cell membrane and membranin, it plays an important role to the cancer of cell and transfer and hemopoietic function.Therefore, will with N-sugar chain for analytic target in the present invention's research.
Up to the present, nucleus magnetic resonance (NMR) technology has become the effective tool that sugar chain stereochemical structure is analyzed.Major advantage is that condition determination is gentle, neither changes structure and the physico-chemical property of sample, can determine again the configuration of sugar, link position, branch and microcosmic diversity.But the fignal center that NMR measures sugar is overlapping serious, and it is more difficult to resolve, and sensitivity is low, and needs milligram level sample, and this is very inaccessible to the micro-sugar chain analysis in most saccharide complex.By comparison comparatively, modern efficient liquid chromatography-mass spectrography (LC-MS) coupling technique utilizes remarkable separating power and the high duplication of liquid technology, the multi-channel testing function of mass-spectrometric technique, particularly high resolution mass spec relies on its universality, highly sensitive and wider dynamic detection range, specific feature, has 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 non-renewable, and there is the problem can not differentiating sugar chain three-dimensional arrangement.The traditional method of analyzing glucoprotein sugar chain is generally sugar chain cut away (hydrazinolysis method and enzyme process) and analyze after separation and purification.Because sugar chain itself does not have chromophoric group, and its not easily ionization on mass spectrograph, in order to more effectively sugar chain can be detected in separation and purification and Structural Identification process, generally carry out the method for column front derivation.The method mainly makes ultraviolet or fluorophor on sugar chain band, improves the sensitivity detected, and can make hydrophobic grouping on sugar chain band again simultaneously, reduces the polarity of sugar chain, sugar chain is retained on reverse-phase chromatographic column, is beneficial to the separation of sugar chain.At present the reagent of derivatize mark carries out to sugar chain more, mainly contain 1-phenyl-3-methyl-5-pyrazolones ketone (PMP), PA (2-AP), Aminobenzoate class derivative reagent (ABEE), phenyl amines derivative reagent (2-AB).Although may be used for after utilizing these reagent to mark sugar chain detecting on fluorescence or MS after HPLC is separated, can can improve selectivity and the sensitivity of sugar chain analysis to a certain extent, but the more difficult control of the chemical reaction condition of these reagent, different sugar chain structure needs to grope different derivatising condition, not only easily there is de-glycosylation phenomenon, and need its formality of multi-step sequence loaded down with trivial details time-consuming.Wherein reductive amination method becomes one of conventional derivatization method due to product stable, but the reducing end of the method to sugar chain directly marks, and the recovery of raw sugar chain is more difficult, also may there is reducing end isomerization in derivatize process, β-eliminative reaction, sialic acid lose and cause the situation occurring during detection being difficult to judge, and the sugar chain after derivative can form reducing end open loop structure, causes the part biological information dropout of sugar chain and impacts some activity of sugar chain.
In recent years, after Kamoda (J.ProteomeRes.4 (2005) 146-152.) etc. utilizes N-Glycosylase F (PNGaseF) enzymolysis to obtain complete N-sugar chain and peptide section, sugar chain reducing end and ammonium salt etc. are reacted and generates intermediate product sugar ammonium (Glycosylamine), sugar ammonium reacts with fluorescence derivatization again, develops N-sugar chain analytical procedure.Although this analytical procedure keeps the original structure of sugar chain in sugar chain derivatize process, the advantage of reducing end open loop can not be formed, but because using fluorescence derivatization, sometimes testing requirement is not reached at LC-MS, and sugar chain structure resolves difficulty, is difficult to reach the object analyzing multiple sugar chain simultaneously.
Summary of the invention
The object of the invention is to overcome existing amido functional group metabolite and the N-sugar chain deficiency in LC-MS detection sensitivity, there is provided a kind of own with positive charge, and the particular fragment ion that mass-to-charge ratio (m/z) is 120 can be obtained in MS/MS, can at electro-spray ionization positive ion (ESI
+) under pattern, mass spectrographic detection sensitivity can be significantly improved, be easy to the mass spectrum derivatization reagent of the amino-functional compounds of Structure Deduction and sugar chain indicia band positive charge.
Technical scheme provided by the present invention is:
A kind of amino-functional compounds and sugar chain indicia band positive charge mass spectrum derivatization reagent, with triphenylphosphine (Triphenylphosphine, TPP) and 2-[4-(2-bromomethylphenyl)]-propionic acid [2-[4-(Bromomethyl) phenyl]-
Propionicacid (2-4-BMPPA)], N-hydroxy-succinamide (N-hydroxysuccinimide) is initiator, Reactive Synthesis [4-(methyl) phenyl-2-propionyl-N-succinimido]-triphenylphosphoniumbromide (MPST), this MPST reagent has following structural formula:
。
The present invention, imagines and carries out the exploitation of chemical derivatization method highly sensitive, highly selective LC-MS analytical procedure to amido functional group metabolite; For N-sugar chain in glycoprotein, first after utilizing N-Glycosylase F (PNGaseF) enzymolysis to obtain complete N-sugar chain and peptide section, sugar chain reducing end and ammonium salt etc. are reacted and generates intermediate product sugar ammonium, sugar ammonium reacts with the mass spectrum derivatization reagent newly synthesized again, exploitation highly sensitive, highly selective N-sugar chain analytical procedure.For biological metabolism group and the further investigation of micro-sugar chain biological function and the screening of various disease biomarkers provides effectively, reliable analyzing and testing means.
The present invention is conceived to positively charged, easily protonated and at electron spray(ES) positive ionization mode (ESI
+) in, being conducive to triphenylphosphine (Triphenylphospine) chemical structure that Selective reaction monitoring (SRM) or multiple-reaction monitoring (MRM) analyze is parent, develop there is highly sensitive, positively charged mass spectrum derivatization reagent that the target amido functional group of highly selective can mark sugar chain simultaneously.The exploitation of positively charged mass spectrum derivatization reagent, by to development and set up highly sensitive, highly selective organism in micro-sugar chain analytical procedure and play bottleneck effect containing amido functional group metabolite analysis method, to the real effective sugar chain biomarker of exploration screening and metabolism biological marker, significant.
The present invention relates to amino-functional compounds and N-connection sugar chain analyrical reagent, specifically refer to MPST reagent.This reagent contains positively charged triphenylphosphine structure, can carry out chemical derivatization reaction to sugar chain and amido functional group metabolite in the glycoprotein of N-Glycosylase F (PNGaseF) enzymolysis; Adopt application of gas chromatorgraphy/mass technology, the amido functional group metabolite and the N-that set up highly sensitive, highly selective link sugar chain simultaneous analysis method.Amido functional group metabolite and the detection sensitivity of sugar chain derivative on mass detector can be improved, and be easy to Structure Deduction.The screening of various disease biomarkers learned for biological metabolism group and otherness sugar group provides effectively, reliable reagents for mass spectrogram.
Below in conjunction with accompanying drawing, the embodiment of the present invention is further described.
Accompanying drawing explanation
Fig. 1 is the mass chromatogram of the LC-MS of embodiment MPST reagent.
Fig. 2 is the mass spectrum of 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.
Embodiment
Embodiment
The synthesis of MPST:
Take MPT85.0mg(0.2mmol) be dissolved in 6.0mL acetonitrile solvent, add DCC(N, N '-dicyclohexylcarbodiimide) 41.2mg(0.2mmol), finally add N-hydroxysuccinimide 23.0mg(0.2mmol) and, stirring at room temperature reacts 16 hours.By reaction solution suction filtration removing white powder, after being spin-dried for solvent, obtain 127.4mg resultant.Adopt eluent methyl alcohol: methylene dichloride=1:25 carries out column chromatography purification.ESI-MS spectral data (
m/z): 425.17 [M]
+;
1h-NMR: δ 1.56-1.59 (d, 3H,
j=9Hz), 2.84 (s, 4H), 3.93-4.00 (dd, H,
j=12.5Hz), 5.31-5.48 (d, 2H,
j=51Hz), 7.07-7.28 (m, 4H), 7.62-7.80 (m, 15H).Fusing point is 128
oc, yield is about 94.9%:
。
As can be seen from the mass chromatogram and mass spectrum of the LC-MS of Fig. 1, Fig. 2 we, the MPST reagent molecule amount of synthesis is consistent with the mass-to-charge ratio m/z=522.18 detected.Can judge that the compound synthesized is MPST reagent really.Fig. 3 specifically represents the MPST reagent of synthesis and the reactive site of amino-functional compounds and reaction structure formula.Fig. 4 specifically represents that N-Glycosylase F (PNGaseF) enzymolysis sialoglycopeptide Sialylglycopeptide (SGP) becomes the process of sugared ammonium structure, and carries out the reaction structure formula of derivative reaction with the MPST reagent of synthesis.
Reagent in the present invention contains positively charged triphenylphosphine structure, because this kind of reagents for mass spectrogram itself are with positive charge, and can at the electro-spray ionization positive ion (ESI of mass spectrometric detection
+) under pattern, can significantly improve determinand Ionization Efficiency and can obtain in MS/MS mass-to-charge ratio (
m/z) be 120 particular fragment ion, detection sensitivity can be significantly improved in Selective reaction monitoring (SRM) or multiple-reaction monitoring (MRM) pattern, and (precursorionscan) can utilize in precursor scans
m/zit is 627 contain
n-Acetylglucos amine structure sugar chain particular child ion, detects parent ion simultaneously, carries out Structure Deduction.With Serine, phenylalanine, the α-amino-isovaleric acid of amido functional group, the detection sensitivity after the derivatizes such as monosaccharide, D-glucose amine (D-Glucamine), sialoglycopeptide Sialylglycopeptide (SGP) is 0.3amol-30fmol.Apparently higher than existing fluorescence derivatization and reagents for mass spectrogram.Chromatography-mass spectroscopy (LC-MS) coupling technique can be utilized, set up the analytical procedure that amido functional group metabolite and N-connect highly sensitive, the highly selective of sugar chain.The screening of various disease biomarkers learned for biological metabolism group and otherness sugar group provides effectively, the reagents for mass spectrogram of highly sensitive, highly selective reliably.
Claims (1)
1. an amino-functional compounds and sugar chain indicia band positive charge mass spectrum derivatization reagent, with triphenylphosphine (Triphenylphosphine, TPP) and 2-[4-(2-bromomethylphenyl)]-propionic acid [2-[4-(Bromomethyl) phenyl]-
Propionicacid (2-4-BMPPA)], N-hydroxy-succinamide (N-hydroxysuccinimide) is initiator, Reactive Synthesis [4-(methyl) phenyl-2-propionyl-N-succinimido]-triphenylphosphoniumbromide (MPST), this MPST reagent has following structural formula:
。
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Cited By (6)
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| CN106770838A (en) * | 2017-02-15 | 2017-05-31 | 延边大学 | Amido functional group chipal compounds split mark band fluorescence derivatization |
| CN106905472A (en) * | 2017-04-18 | 2017-06-30 | 北京蛋白质组研究中心 | A kind of functionalization responsive to temperature type polymer and preparation method and application |
| 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 |
| CN111208254A (en) * | 2019-12-31 | 2020-05-29 | 延边大学 | Derivatization reagent for sugar chain relative quantitative isotope labeling mass spectrum |
| 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 |
| WO2021234002A1 (en) * | 2020-05-20 | 2021-11-25 | F. Hoffmann-La Roche Ag | Reagent for mass spectrometry |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106770838A (en) * | 2017-02-15 | 2017-05-31 | 延边大学 | Amido functional group chipal compounds split mark band fluorescence derivatization |
| CN106770838B (en) * | 2017-02-15 | 2019-05-14 | 延边大学 | Amido functional group chipal compounds split label band fluorescence derivatization |
| CN106905472A (en) * | 2017-04-18 | 2017-06-30 | 北京蛋白质组研究中心 | A kind of functionalization responsive to temperature type polymer and preparation method and application |
| 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 |
| CN111208254A (en) * | 2019-12-31 | 2020-05-29 | 延边大学 | Derivatization reagent for sugar chain relative quantitative isotope labeling mass spectrum |
| CN111208254B (en) * | 2019-12-31 | 2023-09-22 | 延边大学 | Sugar chain relative quantitative isotope labeling mass spectrum derivatization reagent |
| WO2021234002A1 (en) * | 2020-05-20 | 2021-11-25 | F. Hoffmann-La Roche Ag | 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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