Characterization Of Glycopeptides By Stepped NCE On A Hybrid Quadrupole Orbitrap Mass Spectrometer

Protein glycosylation is an important post-translational modification and plays crucial roles in physiological and pathological processes.Glycosylation serves important functions in many biological processes including metabolism and immune regulation.Increasing evidence also demonstrates that aberrant glycosylation is associated with many disease progression such as cancer.Thus,it is of great significance to investigate protein glycosylation.With developments in mass spectrometry-based proteomic field,utilization of mass spectrometry for the characterization of protein glycosylation has achieved great success in the past decades.However,due to the low abundance of glycoproteins and the heterogeneity of glycans,many problems remain to be solved,such as how to effectively enrich glycopeptides from various biological samples and fragment intact glycopeptides to generate information both on glycan structure and peptide sequence.Aiming at these problems,this research work was mainly focused on two parts as follows.1.Evaluating the performance of a new synthetic poly-PD-modified mesoporous silica?ploy-PD@SiO₂?materials for glycopeptides enrichment by using a Hybrid Quadrupole Orbitrap mass spectrometer.Firstly,fetuin and bovine serum albumin?BSA,as an interference protein?were mixed in different ratios to prepare a series of standard samples.Trypsin was then used to digest samples.At last,glycopeptides were enriched by ploy-PD@SiO₂ before MS analysis.With the optimized enrichment procedures and MS analysis conditions,samples enriched by hydrophilic interaction liquid chromatography?HILIC?were mainly non-glycopeptides when resisting 10-fold BSA interference,but ploy-PD@SiO₂ could successfully enrich glycopeptides from samples with resisting up to 100-fold BSA interference.Therefore,we have demonstrated that poly-PD@SiO₂ could provide more effective enrichment for glycopeptides in comparison with HILIC and established a method to evaluate the performance of new synthetic materials.2.A Stepped NCE?Stepped Normalized Collisional Energy?approach for glycopeptides characterization on a Hybrid Quadrupole Orbitrap mass spectrometer.Firstly,intact glycopeptides were enriched from fetuin and RNase B tryptic digests by commercial HILIC.After that,NCEs of 10%,20%,30%,40%and 50%were utilized to cleavage glycopeptides parent ions.Then,fragment behaviors of intact glycopeptides at different collision energies were summarized.The results indicated that NCE of 20%was found to be the optimal parameter for fragmentation of glycan chains,whereas NCE of 30%or 40%resulted in formation of comprehensive peptide fragments.Based on these analysis results,NCEs of 20%,30%and 40%were the optimal NCE setting for stepped NCE analysis of glycopepyides.Thus,abundant fragments of glycan chains and peptide backbones could be captured simultaneously in a single mass spectrum.Finally,the optimized stepped NCE tandem mass spectrometry approach was employed to identify the N-linked glycopeptides enriched from human serum tryptic digests,which demonstrated its utility for characterization of intact glycopeptides in large-scale.In conclusion,a method to evaluate the performance of a new material for glycopeptides enrichment and a stepped NCE approach for studying intact glycopeptides structures were established respectively by using a high resolution and high mass accuracy Hybrid Quadrupole Orbitrap mass spectrometer,which may have wide application prospects in the enrichment and identification of glycoproteins.