Phosphoproteome Profiling Using Immobilized Fe(Ⅲ) Affinity Chromatography with Mass Spectrometry

• Main Authors: LAI CHEN-YU, 賴承煜
• Other Authors: CHEN YU-JU
• Format: Others
• Language: en_US
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/82245048860080562486

碩士 === 國立臺灣師範大學 === 化學系 === 93 === The protein phosphorylation is key step in cellular signaling to initiate various cellular functions. Despite the advances of various powerful analytical methods is available, global characterization of the site specific phosphorylation remains far from routine practice. The characterization of phosphorylation has been challenged by the technical difficulty associated with their abundance in cell, dynamic modification pattern, and heterogeneous forms of phosphoproteins. The challenge warrants the need to develop methods capable of accurately elucidating sites of protein phosphorylation. In this study, a high throughput platform combining subcellular fractionation, SDS-PAGE, immobilized metal affinity chromatography (IMAC) and mass spectrometry (MS) was introduced for the analysis of phosphoprotein form complex protein mixture.The loading capacity, binding specificity, sample recovery, elution volume and elution reagent of the IMAC were optimized and studied in detail. To demonstrate the feasibility for the analysis of complex protein mixture, the phosphopeptide from the crude protein extract of the human jurkat T-cell was subjected to the SDS-PAGE separation, IMAC purification, followed by the nLC-ESI-MS analysis. The preliminary result identified 782 phosphopeptides with 891 phosphorylation sites from 603 proteins in cytosolic fraction, about 90% of the identified peptides were found to be phosphorylated using optimized IMAC parameters. This technology platform can be applied to profiling protein phosphorylation on the proteome level.