An Unambiguous Method For Identification Of Protein Phosphorylation And Phosphoproteomic Analysis Of The HT1080 Cell

Protein phosphorylation is one of the most importantpost-translationalmodifications in cells, and the reversiblephosphorylation of proteins regulates nearly all aspects ofcell life,So to accurately elucidate phosphorylation sites ofproteins is the first task and the key procedure inphosphoproteomics research. In this paper, we have developed areliable strategy that combines immobilized metal affinitymagnetic beads chromatography with tandom mass spectrometry toanalyze phosphosrylation sites of proteins.In chapter one, a simple method was established foraccurately, rapidly identification of phosphorylation sitesfrom theβ-casein and synthesized phosphopeptides:thephosphopeptides were firstly selectively enriched byimmobilized metal affinity beads chromatography followed by4-sulfophenyl isothiocyanate(SPITC)derivatization,then thederivatized phosphopeptides and the phosphorylation sites ofwhich were correctly identified from the y series ions of theirmass spectrum. A different characteristics of the tyrosine andserine phosphorylation type have been disccused, and thespecific m/z peaks information were also summarized for thedefing different types of phosphorylation.In chapter two, we coupled immobilized metal affinitychromatography with MALDI-TOF/TOF mass spectrometry to analyzephosphoproteins in human fibrosarcoma cell line HT1080. A totalof 43 phosphopeptides were observed, 11 phosphopeptide sequences were determined, 11 phosphorylation sitescorresponding to 10 phosphoproteins were defined. All thephosphoproteins identifided from the cell line are categorizedaccording to their functions and the localization of theseproteins.