A comprehensive identification method for the primary structure of intact n-glycoprotein

Abstract

The invention relates to a method for completely identifying a primary structure of complete N-glycoproteins. The method comprises the following steps: sample pretreatment of N-glycoproteins., namelyreacting a glycoprotein solution and a DTT (Dithiothreitol) solution, and diluting with an electrospray buffer solution; loading samples by a syringe and a syringe pump, spraying in a positive ion mode, and collecting a primary spectrum and a secondary spectrum of the samples by an orbitrap mass spectrometer based on HCD (Host Controller Driver) dissociation; generating an N-glycoprotein precursorion theory database; generating an N-polysaccharide precursor ion theory database; analyzing an N-polysaccharide topological structure; analyzing the N-glycoproteins structure. Compared with the prior art, the method disclosed by the invention has the advantages that the polysaccharide topological structure and the protein structure can be simultaneously detected on a complete protein level, andselectively dissociated characteristic fragment ions of the polysaccharides are obtained. The method is simple in step and high-efficiency and accurate in identification. The method is applicable to polysaccharide topological structure identification, glycosylation site determination and protein scaffold analysis of glycoproteins based on high-resolution tandem mass spectrometry.

Description

technical field [0001] The present invention relates to technical fields such as systems biology and glycoproteomics related to biological mass spectrometry, and in particular relates to a method. Background technique [0002] Glycosylation is one of the most common and important post-translational modifications on proteins. More than 50% of proteins in the human body are glycosylated, and glycosylation has extremely important physiological and pathological functions (Rudd, P.M.; Elliott, T.; Cresswell, P.; Wilson, I.A.; Dwek, R.A., Glycosylation and the immune system. Science 2001, 291(5512), 2370-2376.). Glycoproteomics based on tandem mass spectrometry has become one of the important analytical methods for the identification and analysis of protein amino acid sequence, protein glycosylation site, monosaccharide composition and topological structure ((a) Raman, R.; Raguram, S .; Venkataraman, G.; Paulson, J.C.; Sasisekharan, R., Glycomics: an integrated systems approach t...

AI Technical Summary

Problems solved by technology

The above two methods are cumbersome and time-consuming, and are very prone to missed cuts; the obtained glycoprotein structure information is also limited

Since the polysaccharide structure modified by protein glycosylation has many connections and branched isomers, there is great structural heterogeneity, which brings great difficulties to characterize its structure in depth; other studies have shown that glycosylation affects the stability of proteins Also has an effect, the amino acid sequence around the glycosylated modification site is more difficult to break than the same sequence without glycosylation (for example, RNase B and RNase A) (Bourgoin-Voillard, S.; Leymarie, N.; Costello, C.E., Top-downtandem mass spectrometry on RNase A and B using a Qh / FT-ICR hybrid massspectrometer. Proteomics 2014, 14(10), 1174-1184.), so it is also important for the characterization of the protein backbone and the positioning of glycosylation sites raised a challenge

Images