A phosphohistidine proteomics strategy based on elucidation of a unique gas-phase phosphopeptide fragmentation mechanism.
A phosphohistidine proteomics strategy based on elucidation of a unique gas-phase phosphopeptide fragmentation mechanism.
复制标题
DOI：
10.1021/ja507614f
复制
发表时间：
2014-09-17
影响因子：
15
通讯作者：
Muir, Tom W.
中科院分区：
文献类型：
--
作者：
Oslund, Rob C.;Kee, Jung-Min;Couvillon, Anthony D.;Bhatia, Vivek N.;Perlman, David H.;Muir, Tom W.
蛋白质组氨酸磷酸化是中枢代谢和细胞信号转导的关键翻译后修饰(PTM)。尽管如此，由于缺乏丰富和鉴定这种不稳定的PTM的工具，在蛋白质组中检测磷酸组氨酸(PHI)仍然很困难。为了解决这个问题，我们报道了第一个关于PHIS蛋白的全球蛋白质组分析，结合了PHIS多肽的选择性免疫浓缩和基于对PHIS多肽在LC-MS/MS期间的气相碎裂的机理洞察的生物信息学策略。我们表明，PHIS多肽的碰撞诱导解离(CID)产生了显著的特征中性损失98，80和116 Da。利用同位素标记研究，我们还证明了98Da的中性损失是通过气相磷酸转移到肽C-末端的α-羧酸盐或转移到谷氨酸/天冬氨酸侧链残基上发生的。为了利用这一特性，我们开发了一个软件工具，根据含有PHIs的多肽的中性损失模式筛选LC-MS/MS光谱，以寻找潜在的匹配。该工具被集成到蛋白质组学工作流程中，用于鉴定细胞裂解物中含有PHI的内源性蛋白质。作为这一策略的一个例证，我们分析了甘油和甘露醇喂养的大肠杆菌细胞中的PHIs多肽。我们确定了一些已知的和以前推测的PHI位点，主要是在磷酸烯醇式丙酮酸：糖转移酶系统(PTS)中。此外，我们在乙醛-乙醇脱氢酶(ADHE)和丙酮酸激酶(PYKF)酶上发现了两个新的组氨酸磷酸化位点，这两个位点以前从未被发现具有这种修饰。这项研究为未来在细菌和其他生物中进行PHI蛋白质组学研究奠定了基础。
Protein histidine phosphorylation is increasingly recognized as a critical posttranslational modification (PTM) in central metabolism and cell signaling. Still, the detection of phosphohistidine (pHis) in the proteome has remained difficult due to the scarcity of tools to enrich and identify this labile PTM. To address this, we report the first global proteomic analysis of pHis proteins, combining selective immunoenrichment of pHis peptides and a bioinformatic strategy based on mechanistic insight into pHis peptide gas-phase fragmentation during LC–MS/MS. We show that collision-induced dissociation (CID) of pHis peptides produces prominent characteristic neutral losses of 98, 80, and 116 Da. Using isotopic labeling studies, we also demonstrate that the 98 Da neutral loss occurs via gas-phase phosphoryl transfer from pHis to the peptide C-terminal α-carboxylate or to Glu/Asp side chain residues if present. To exploit this property, we developed a software tool that screens LC–MS/MS spectra for potential matches to pHis-containing peptides based on their neutral loss pattern. This tool was integrated into a proteomics workflow for the identification of endogenous pHis-containing proteins in cellular lysates. As an illustration of this strategy, we analyzed pHis peptides from glycerol-fed and mannitol-fed Escherichia coli cells. We identified known and a number of previously speculative pHis sites inferred by homology, predominantly in the phosphoenolpyruvate:sugar transferase system (PTS). Furthermore, we identified two new sites of histidine phosphorylation on aldehyde-alcohol dehydrogenase (AdhE) and pyruvate kinase (PykF) enzymes, previously not known to bear this modification. This study lays the groundwork for future pHis proteomics studies in bacteria and other organisms.
登录
查看更多内容
影响因子：
46.9
作者：
Rush, J;Moritz, A;Comb, MJ
通讯作者：
Comb, MJ
影响因子：
4.8
作者：
Morgan, Hugh P.;McNae, Iain W.;Walkinshaw, Malcolm D.
通讯作者：
Walkinshaw, Malcolm D.
影响因子：
8
作者：
Medzihradszky, KF;Phillipps, NJ;Turck, CW
通讯作者：
Turck, CW
影响因子：
4.8
作者：
Cai, SJ;Inouye, M
通讯作者：
Inouye, M
影响因子：
46.9
作者：
Ficarro, SB;McCleland, ML;White, FM
通讯作者：
White, FM