Development of Technology and Method for Phosphoproteome and Glycoproteome Analysis

The elucidation of protein post-translational modifications, such as phosphorylation, remains a challenging analytical task for proteomic studies. Since many of the proteins targeted for phosphorylation are low in abundance and phosphorylation is typically substoichiometric, a prerequisite for their identification is the specific enrichment of phosphopeptide prior to mass spectrometric analysis. A new type of the immobilized metal ion affinity chromatography (IMAC) through the chelating interaction between phosphate groups on the polymer and Zr^4＋ and Ti^4＋ (Zr^4＋- and Ti^4＋-IMAC) has been developed for enriching phosphopeptides. We also compared Zr^4＋- and Ti^4＋-IMAC to other enrichment methods including Fe^3＋-IMAC, TiO₂ and ZrO₂, and demonstrate superior selectivity and efficiency of Zr^4＋- and Ti^4＋-IMAC for the isolation and enrichment of phosphopeptides. Furthermore, a new approach was established by integration of the enrichment of phosphopeptides with Ti^4＋-IMAC and peptide fractionation with strong cation-exchange (SCX) column for large-scale phosphoproteome analysis of human liver, and about 10 000 phosphorylation sites were detected. Compared with large scale phosphorylation analysis at proteome level, comprehensive and reliable phosphorylation site mapping of individual phosphoprotein is equally important. A novel method for confident phosphorylation site analysis of individual phosphoproteins was developed without manual interpretation of spectra. Phosphopeptides enriched from tryptic digests of phosphoproteins were analyzed by nano-LC-MS²/MS³, and the acquired MS² and MS³ spectra in valid MS²/MS³ pairs were searched against the composite database separately. This methodology combined with multi-protease digestion approach was applied to analyze the phosphorylation of the standards phosphoproteins and cyclic AMP dependent protein kinases.In silico digestion of proteins in human proteome database by trypsin indicates that a significant percentage of tryptic N-glycopeptides is not in the preferred detection mass range of shotgun proteomics approach, that is, from 800 to 3 500 Da. And the quite big size of glycan groups may block trypsin to access the K, R residues near N-glycosites for digestion, which will result in generation of big glycopeptides. Thus many N-glycosites could not be localized if only trypsin was used to digest proteins. We developed a comprehensive way to analyze the N-glycoproteome of human liver tissue by combination of hydrazide chemistry method and multiple enzyme digestion, which leads to great improvement in coverage of N-glycosites identified.