Accurate Determination Of Glycoprotein Molecular Weight And Study Of Protein Interactions Based On Mass Spectrometry

Glycoprotein is one of the most common post-translational modifications.It participates in most of the life processes of the human body.Most proteins in the organism perform their functions mainly through direct contact.Changes in glycosylation patterns can cause various diseases,including cancer,inflammation,etc.,and have a certain impact on the safety and efficacy of therapeutic recombinant glycoproteins.Therefore,characterizing the interaction between glycoproteins is helpful to discover the role of glycoproteins in cellular physiological processes.In order to analyze glycoprotein complexes under near-physiological conditions and accurately determine the binding ratio and interaction relationship of the complexes,it is necessary to accurately determine the molecular weight distribution and glycosylation modification degree of the intact glycoproteins that make up the complex.For the glycoprotein with a determined degree of modification,the modification sites and other modification sites related to the structure and function of the sequence are further accurately characterized.Therefore,the analysis of glycoproteins and their complexes is based on the levels of intact glycoprotein complexes,intact glycoproteins and glycosylated peptides respectively.Analyzing glycoproteins based on the intact protein level,if the degree of glycosylation is high,the adjacent valence peaks of the intact protein overlap,and the spectrum is complicated,and it is difficult to identify its exact molecular weight.The use of charge reduction technology can improve the separation between adjacent valence peaks,simplify the spectrum,and improve the accuracy of the molecular weight determination of complex glycoprotein systems.For different glycoprotein systems,we mainly evaluated the accuracy of different charge reduction techniques for the determination of glycoprotein molecular weight from the separation between adjacent valence peaks and adjacent glycoform peaks,in order to select a suitable charge reduction method for accurate determination.The molecular weight and degree of glycosylation of different glycoprotein systems have certain reference significance.Monoclonal antibodies are also a common glycoprotein drug.In order to accurately profile the glycoforms of the monoclonal antibody directly from the intact glycoprotein level,we explored the influence of various factors on the identification of glycoprotein glycoforms,and realized the optimization of the conditions for accurate determination of the glycoforms of the monoclonal antibody.In order to further obtain the detailed characterization of the sequence,modification and conformation of the monoclonal antibody,we plan to develop an ultra-high resolution mass spectrometry method based on the top-down strategy.At the same time,we use capillary electrophoresis technology as a front-end separation method.The capillary can be used as a reactor for online reaction.The artificial control of the reduction degree of the monoclonal antibody has been realized,and the structure of the monoclonal antibody has been simplified,which is conducive to the top-down analysis of the monoclonal antibody.Based on the analysis of the interaction relationship between glycoproteins at the level of glycoprotein complexes,in order to explore the heat-induced agglutination products of the highly glycosylated protein Haptoglobin,we combined size exclusion chromatography with non-denaturing mass spectrometry to prove that Haptoglobin heat-induced agglutination The product is different from the agglutination product formed by thermal transformation of most proteins and can be spontaneously decomposed.In order to extend the non-denaturing mass spectrometry technology to the application of non-glycosylated protein interactions,we explored the information transmission mechanism of protein interaction during the activation of the innate immune pathway,and obtained the changing trend of the binding and quantitative relationship of protein complexes under near-physiological conditions.