A Label-free LC/MS Strategy for Quantitative Profiling of Phosphoproteome

• Main Authors: YI-TING WANG, 王薏婷
• Other Authors: Kuo-Lung Ku
• Format: Others
• Language: en_US
• Online Access: http://ndltd.ncl.edu.tw/handle/50633939387038954560

碩士 === 國立嘉義大學 === 應用化學系研究所 === 96 === Protein phosphorylation plays a critical role in normal cellular functions and abnormal phosphorylation is often associated with disease. Despite its importance, qualitative and quantitative analysis of site-specific protein phosphorylation presents an analytical challenge due to its heterogeneity and low abundance at proteome scale. Towards comprehensive and robust multiplexed quantification for phosphoproteome, we developed a label-free quantification strategy integrating optimized gel-assisted digestion with a highly specific and reproducible automatic pH-acid controlled IMAC, followed by LC-MS/MS analysis. By four-dimensional alignment algorithm, termed SEMI, on Sequence, Elution time, Mass to charge and Internal standard, the variations between different LC-MS/MS runs can be corrected, thus increasing the quantifiable numbers of phosphoproteins. Without conventional isotopic labeling, we used the integrated chromatographic peak areas of phosphopeptide for quantification. By addition of internal standard protein before gel-assisted digestion, the quantitation deviation of phosphopeptides during tryptic digestion, IMAC purification and LC-MS/MS can be normalized. The reliable linear correlation can be obtained with 4000-fold dynamic concentrations (R2=0.99) for standard phosphoprotein, ovalbumin. By SEMI alignment approach, quantifiable numbers of phosphopeptides increase from 331 to 1679 with 14% of standard deviation1 in quantification, demonstrating a sensitive, accurate and stable quantification platform for phosphoproteomics. In the second part of thesis, we presented the applications of the new approach on two systems: (1) Altered tyrosine phosphorylation on cell membrane upon stimulation of pervanadate. (2) Comparative analysis of phosphoproteome in four lung cancer cell lines with different invasiveness potential. Pervanadate is a powerful protein tyrosine phosphatase inhibitor that is commonly used to maintain tyrosine phosphorylation in cells. Using the optimized gel-assisted digestion and IMAC purification, we performed comparative study on tyrosine phosphorylation on total cell lysate and on cell membrane. We identified 185 and 341 phosphotyrosine peptides with percentage of 8% and 20% for total cell lysate and cell membrane, respectively. The results demonstrated that pervanadate indeed increases the percentage of phosphotyrosine in physiological conditions. The second application was demonstrated on the quantitative profiling of phosphoproteome in lung cancer cell with varying degree of invasiveness. Lung cancer is one of the most common cancers in the world and the overall five-year survival rate is 14%, and most patients in whom therapy fails have distant metastases. Recently, phosphorylation has been reported to be required in metastases. Analysis on the four lung adenocarcinoma cell lines identified a total of 1231 phosphoproteins and 2525 phosphopeptides were quantifiable. A significant number of proteins (838) were found to be differentially phosphorylated in metastatic lung cancer cells compared to the one without invasive potential. Some of these constituently phosphorylated and over-activated protein kinases have been known to involve in lung adenocarcinoma, including ERK/MAPK signaling pathway and cell migration. We conclude that this platform provides a generic and powerful complement for phosphoproteomics to identify abnormal phosphoprotein associated with disease. With the advantages of multiplexed quantification, high sensitivity and cost-effective strategy, it may present a useful approach to decipher the dynamically and spatially heterogeneous phosphoproteome.