A method for relative quantification of n-glycopeptide-terminal sialic acid α2,6 and α2,3 linked isomers

A terminal sialic acid, relative quantitative technology, applied in the field of analytical chemistry, can solve the problems of lack of convenient, fast and environmentally friendly quantitative methods, limited analysis of polysaccharide peptide samples, inability to use glycopeptide sialic acid α2, etc., to achieve accurate quantification. High degree, high accuracy, environment optimization effect

Active Publication Date: 2022-05-20
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method cannot be used for quantitative analysis of glycopeptide sialic acid α2,3- / α2,6-linkage isomerism
PGC separation and reversed-phase chromatography separation at high column temperature can also achieve the separation of some glycopeptide sialic acid linkage isomers, but the analysis of complex polysaccharide peptide samples is limited
Therefore, there is a lack of convenient, fast and environment-friendly quantitative methods for the relative quantitative analysis of terminal sialic acid α2,3 / α2,6 linkage isomerism of complex N-glycopeptides

Method used

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  • A method for relative quantification of n-glycopeptide-terminal sialic acid α2,6 and α2,3 linked isomers
  • A method for relative quantification of n-glycopeptide-terminal sialic acid α2,6 and α2,3 linked isomers
  • A method for relative quantification of n-glycopeptide-terminal sialic acid α2,6 and α2,3 linked isomers

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Experimental program
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Embodiment 1

[0022] All chemical reagents and solvents used in the examples are analytically pure.

[0023] (1) Standard N-glycopeptide sample preparation: Dissolve standard intact N-glycopeptides (α2,6-SGP and α2,3-SGP) linked to terminal sialic acid α2,6 and α2,3 in 0.1 % TFA to obtain two standard stock solutions at a final concentration of 8.7 μM. Standard glycopeptides α2,6-SGP and α2,3-SGP were mixed in absolute concentration (87 nM) in different mixing ratios (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1) , prepared as a sample for analysis.

[0024] (2) Liquid phase separation of mixed standard glycopeptides: On-line LC uses the M-Class nano-LC system of Waters Company: Phase A is 0.1% FA aqueous solution; phase B is ACN solution with 0.1% TFA. N-glycopeptides were loaded on-column at a flow rate of 300 nL / min for 4 min. Then it enters the analytical column for separation, and the chromatographic column adopts 15 cm C 18 Column (nanoE MZ PST CSH130 C 18 1.7μ75μm × 150mm...

Embodiment 2

[0030] Using haptoglobin (Hp) purified in serum as an analyte, the complete N- Relative proportion of α2,3 and α2,6 isomeric linkages of glycopeptide terminal sialic acids.

[0031] (1) Purification of serum haptoglobin: Serum samples were centrifuged at 12000*g for ten minutes, and then purified using HiTrap columns. The haptoglobin bound to the column was eluted with elution buffer (pH 3.0, 100 mM Glycine, 0.5 M NaCl), followed by acetone precipitation and concentration.

[0032] (2) Enzymatic hydrolysis of haptoglobin and purification of N-glycopeptide: Take 190 μl of haptoglobin with a concentration of 1 mg / mL, add 10 μl of 200 mM dithiothreitol (DTT) solution, react at 56 °C for 1 h, and perform protein Denaturation and reduction treatment. Then add 10ul of 400mM iodoacetamide solution (IAA), and react in the dark for 30 min at room temperature to carry out the alkylation treatment of the protein. Finally, 4 μg of trypsin was added for enzymatic hydrolysis of the prote...

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Abstract

The invention belongs to the technical field of analytical chemistry, and specifically relates to a method for relative quantification of N-glycopeptide terminal sialic acid α2,6 and α2,3 linkage isomerism. In the present invention, liquid chromatography and ion mobility mass spectrometry are combined, and after complex N-glycopeptide samples are separated by liquid chromatography, parent ions are selected according to specific retention time and specific mass-to-charge ratio, and the collision-induced dissociation under specific conditions , all generated fragments undergo two-dimensional separation in an ion mobility cell and then enter a mass spectrometer for detection; contain terminal sialic acid α2,6 and α2,3 isomer B 3 + The fragments are separated in the flow tube and finally according to their terminal sialic acid α2,6 and α2,3 isomer B 3 + Relative quantification of α2,6 and α2,3 linkage isomerization of N-glycopeptide terminal sialic acid is carried out by peak area of ​​fragment mobility separation peak. The method of the invention is efficient, fast and environmentally friendly, and has broad application prospects in the fields of proteomics, sugar chemistry and biology.

Description

technical field [0001] The invention belongs to the technical field of analytical chemistry, and in particular relates to a relative quantitative method for α2,6 and α2,3 linkage isomerism of N-glycopeptide terminal sialic acid. Background technique [0002] Glycosylation is an important protein post-translational modification (PTM) that exists widely in organisms and has a very important impact on the chemical and biological properties of proteins. On human glycoproteins, sialic acid is an acidic nine-carbon sugar family, which is linked to galactose (Gal) residues through α2,3- or α2,6 glycosidic bonds, and it can serve as a regulatory factor for attachment proteins, It can also be used as a recognition target for other glycoproteins, thereby regulating various physiological and pathological processes. There are various methods for analyzing the linkage isomerism of sialic acid. Traditional spectroscopic methods mainly include NMR and infrared absorption spectroscopy. The...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/72G01N30/86
CPCG01N30/02G01N30/06G01N30/7233G01N30/8675
Inventor 魏黎明封晓晓陆豪杰
Owner FUDAN UNIV
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