In Situ Sample Processing Approach (iSPA) for Comprehensive Quantitative Phosphoproteome Analysis

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• 作者：Junfeng Huang ; Hongqiang Qin ; Jing Dong ; Chunxia Song ; Yangyang Bian ; Mingming Dong ; Kai Cheng ; Fangjun Wang ; Deguang Sun ; Liming Wang ; Mingliang Ye ; Hanfa Zou
• 刊名：Journal of Proteome Research
• 出版年：2014
• 出版时间：September 5, 2014
• 年：2014
• 卷：13
• 期：9
• 页码：3896-3904
• 全文大小：444K
• ISSN：1535-3907

文摘

Current sample preparation protocols for quantitative phosphoproteome analysis are tedious and time-consuming. Here, a facile in situ sample processing approach (iSPA) is developed by using macroporous Ti(IV)-IMAC microspheres as the preparation 鈥渂eds鈥? where all sample preparation procedures including the enrichment of phosphoproteins, tryptic digestion of proteins, enrichment, and isotope labeling of phosphopeptides are performed in situ sequentially. As a result of the in situ processing design and the seamless procedures, extra steps for desalting and buffer exchanging, which are always required in conventional approaches, are avoided, and the sample loss and contamination could be greatly reduced. Thus, better sensitivity and accuracy for the quantitative phosphoproteome analysis were obtained. This strategy was further applied to differential phosphoproteome analysis of human liver tissues with or without hepatocellular carcinoma (HCC). In total, 8548 phosphorylation sites were confidently quantified from three replicate analyses of 0.5 mg of human liver protein extracts.

Keywords:

in situ sample processing approach; phosphoprotein enrichment; on-beads digestion; dimethyl labeling; solid phase labeling; phosphoproteome quantification; human liver; hepatocellular carcinoma