Sequencing of phosphopeptides sulfonated by 4-sulfophenyl isothiocyanate using post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry  

作　　者：TANG Jianguang WANG Yongjun ZHANG Hainan WANG Chunyu HU Zhiping XUE Zhigang XIA Kun SHI Xiaoliu 

机构地区：[1]Department of Neurology, the Second Xiangya Hospital, Central South University, Changsha 410011, China [2]Department of Digestion, the Second Xiangya Hospital, Central South University, Changsha 410011, China [3]National Key Laboratory of Chinese Genetics, Changsha 410078, China

出　　处：《Progress in Natural Science:Materials International》2006年第9期930-935,共6页自然科学进展·国际材料（英文版）

基　　金：Supported by National Natural Science Foundation of China (Grant Nos. 30470619 and 30400537)the Major State Basic Research Development Program of China (2004CB518800)

摘　　要：Phosphorylation/dephosphorylation is probably the most common and important reversible post-translational modification of proteins. Analyzing the functional effects of phosphorylation is helpful for understanding the biological functions of proteins. Identification of the phosphorylation sites of phosphorylated protein is a prerequisite for research on phosphorylation. In this work, an effective and simple method of identification of protein phosphorylation sites has been developed. Phosphopeptides were selectively enriched with immobilized metal affinity chromatography （IMAC） and subsequently chemically modified by 4-sulfophenyl isothiocyanate, and then the chemically modified phosphopeptides were sequenced with post-source decay （PSD） matrix-assisted laser desorption/ionization （MALDI） time-of-flight mass spectrometry for detecting phosphorylation sites. The charge of derivatization by 4-sulfophenyl isothiocyanate introduces a negative sulfonic acid group at the N-terminus of a peptide, and enables the selective detection of only a single series of C-terminal y-type ions. This chemically assisted method greatly simplifies the extremely complex pattern of PSD fragment ions and makes the PSD spectra more easier to be interpreted. The phosphorylation sites of a synthesized model phosphopeptide and human c-myc protein have been successfully identified by this method.Phosphorylation/dephosphorylation 可能是蛋白质的最普通、重要的可逆 translational 以后修正。分析 phosphorylation 的功能的效果对理解蛋白质的生物功能有用。phosphorylated 蛋白质的 phosphorylation 地点的鉴定是为关于 phosphorylation 的研究的一个前提。在这个工作，蛋白质 phosphorylation 地点的鉴定的一个有效、简单的方法被开发了。Phosphopeptides 有选择地与使不能调动的金属亲密关系层析(IMAC ) 被充实并且化学上随后由 4-sulfophenyl isothiocyanate，然后化学上修改的 phosphopeptides 修改了与来源以后的腐烂(PSD ) 被定序为检测 phosphorylation 地点的帮助矩阵的激光解吸附作用 / 电离(MALDI ) time-of-flight 团 spectrometry。由 4-sulfophenyl isothiocyanate 的 derivatization 的费用在肽的 N 终点介绍一个否定的酸性硫酸基的酸组，并且启用仅仅 C 终端 y 类型离子的一个单个系列的选择察觉。这个化学上帮助的方法极大地简化 PSD 碎片离子的极其复杂的模式并且使 PSD 系列更更容易被解释。综合模型 phosphopeptide 和人的 c-myc 蛋白质的 phosphorylation 地点被这个方法成功地识别了。

关 键 词：PHOSPHOPEPTIDES SEQUENCING 4-sulfophenyl isothiocyanate post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. 

分 类 号：Q516[生物学—生物化学]