Mass Spectrometry Following Mild Enzymatic Digestion Reveals Phosphorylation of Recombinant Proteins in Escherichia coli Through Mechanisms Involving Direct Nucleotide Binding

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• 作者：Yi-Min She ; Xiaohui Xu ; Alexander F. Yakunin ; Sirano Dhe-Paganon ; Lynda J. Donald ; Kenneth G. Standing ; Daniel C. Lee ; Zongchao Jia ; Terry D. Cyr
• 刊名：Journal of Proteome Research
• 出版年：2010
• 出版时间：June 4, 2010
• 年：2010
• 卷：9
• 期：6
• 页码：3311-3318
• 全文大小：398K
• 年卷期：v.9,no.6(June 4, 2010)
• ISSN：1535-3907

文摘

A straightforward method using mild enzymatic digestions combined with MALDI mass spectrometry (MS) was used to enhance determination of the multiple phosphorylation sites of a set of recombinant nucleotide-binding proteins in Escherichia coli, including kinases and cystathionine beta-synthase (CBS) domain containing proteins. The protein kinases reveal abundant phosphorylations in the kinase domains and relatively low phosphogluconoylation (258 Da) at the N-terminal His-tag. In contrast, the CBS domain-containing proteins possess a highly conserved phosphorylation in vivo at Ser-2 of the His-tag. Multistage MS/MS and selected reaction monitoring established that the CBS domain proteins also contain a combined modification of gluconoylation (178 Da) and phosphorylation (80 Da) at two different sites, instead of an isobaric phosphogluconoylation (258 Da) event at the N-terminus. Functional analysis of 20 recombinant proteins as identified by mass spectrometry has shown the phosphorylation at the N-terminal His-tag is relevant to nucleotide binding and phosphotransfer reaction catalyzed by a serine protein kinase.