Optimization And Application Of Label-free Quantitative Proteomics And Glycoproteomics

Label free quantification(LFQ)based on mass spectrometry is playing an increasingly important role in biology and clinical research due to simple sample preparation,high throughput and accurate quantification.Glycosylation is a very important post translation modification of proteins.Changes of protein,protein glycosylation and glycan can be illustrated from the perspectives of quantitative protemics,glycoproteomics and glycomics.Based on the demand for deep coverage and high-throughput quantitative proteomics,data independent acquisition(DIA)based label-free quantification showed as a promising method.This study systematically optimized and applied DIA quantitative method to proteome of cell line and plasma.Then,quantitative proteomics,glycoproteomics,and glycomics were integrated to illustrate important roles of glycosylation in paclitaxel(PTX)resistance mechanism in breast cancer cell MCF7.The main conclusions were as follows:(1)The performance of seven spectral libraries based on three different strategies was systematically evaluated and compared.Spectral library generation by DDA analysis of mixed digested peptides from multiple cell lines and high p H reversed phase fractionation to 18 fractions was an effective method balancing mass spectrometry measuring time and spectral library coverage.The spectral library contained more than 150 000 peptides and 9 900 proteins.Using this spectral library,7 047 proteins were quantified by DIA in three technical replicates,covering many proteins involved in signal transduction pathways,low-abundant kinases and transcription factors.Compared to DDA,DIA showed deeper proteome coverage,better data completeness and better quantitative accuracy and precision.(2)To develop a deep,high-throughput and estimated absolute quantitative plasma proteomics workflow,we optimized plasma spectrum library,LC and DIA parameters,and assessed the accuracy of absolute quantification.Nearly 600 plasma proteins with a dynamic range greater than 6 orders of magnitude and LOQ of 13.8 ng/m L were absolutely quantified in a single DIA analysis with a deep plasma spectral library(containing 55 157 peptides and 5 328proteins),50 cm column,90 min gradient,and 26 internal proteins assisted absolute quantification method.(3)The DIA quantitative performance of Q Exactive Plus,Q Exactive HF-X and Orbitrap Fusion Lumos was evaluated in the following study.We established the corresponding DIA method for each mass spectrometer and assessed with Hela QC peptides,high abundant proteins depleted plasma sample,and mixture of yeast and plasma peptides with known ratios.The results show that use of a deep coverage spectral library,resolution of MS2 at 17.5K and the maximum ion injection time of 45 ms,enabled Q Exactive Plus to achieve the comparable DIA performance to advanced mass spectrometer Q Exactive HF-X or Orbitrap Fusion Lumos including comparable number of proteins and peptides,precision and accuracy of quantification.The normalization method of protein intensities could eliminate the batch effect caused by the inconsistent response among mass spectrometers and enable integrative analysis of DIA data from different mass spectrometers with high reproducibility.(4)Integrative strategies of transcriptomics,proteomics,glycomics and glycoproteomics were used to systematically characterize the resistance of MCF7-PTX cells.Total 7 006 proteins were quantified and 2 848 differentially expressed proteins were found through quantitative proteomics.Total 1 358 glycopeptides corresponding to 848 glycoproteins were quantified and150 glycopeptides were differentially expressed through quantitative glycoproteomics.27 different N-glycan structures were quantified and 20 N-glycans were differentially expressed(13 N-glycans were down-regulated and 7 N-glycans were up-regulated)through quantitative glycomics,and most of which were marked as complex glycans with multi-antenna branch and core fucosylation.The results showed that MGAT4 A and its responsible multi-antenna branch structure were down-regulated in MCF7-PTX cells.Glycoprotein MTUS1 had a reduced level of multi-antenna branch structure,and its related ERK signaling pathway was inhibited in MCF7-PTX cells.Therefore,it was proposed that the multi-antenna branch structure may be involved in chemoresistance by regulating the function of the target proteins and related signal pathways.