Investigations On The Separation And Enrichment Of N-glycosylated Proteins And Tyrosine Phosphorylated Proteins

Glycosylation and phosphorylation are two kinds of important protein post-translational modifications,while proteins with such post-translational modifications are of extremely low abundance in complex biological system,and their detection signals in mass-spectroscopy are usually heavily suppressed by the signals of highly abundant proteins/peptides.Therefore,it is quite necessary to develop some simple,efficient and specific methodologies for the separation/enrichment of protein/peptides after post-translational modifications.By using home-made C18 solid phase extraction columns and high pH reversed phase chromatography technique,the separation of protein species in human urine are achieved.A total number of 2550 protein species are identified by using liquid chromatography-mass spectrometry(LC-MS)analysis.At the same time,a novel enrichment protocol is developed for the preconcentration of N-glycosylated protein/peptide human urine with a triblock hydrophilic copolymer grafted silica microparticle(HILIC)as the adsorbent.A total number of 1331 N-glycosylated peptides are identified in human urine,corresponding to 590 N-glycosylated proteins.An efficient tandem enrichment strategy for large scale identification of tyrosine-phosphorylated proteins in mouse liver is also successfully established.The phosphorylated proteins in mouse liver are firstly separated and enriched by the combination of TiO2 affinity enrichment and C18 reversed phase chromatography separation technique,thereafter,tyrosine-phosphorylation antibodies are adopted to specifically enrich the tyrosine-phosphorylated proteins/peptides from the enriched phosphorylated proteins.Mass-spectrometry analysis indicate that a total number of 1297 phosphorylation peptides are identified after the enrichment by TiO2and 916 tyrosine-phosphorylated peptides are identified after the enrichment by tyrosine-phosphorylation antibodies,which is 70.62%of the total identified phosphorylated peptides.