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  • Title: Mass spectrometry following mild enzymatic digestion reveals phosphorylation of recombinant proteins in Escherichia coli through mechanisms involving direct nucleotide binding.
    Author: She YM, Xu X, Yakunin AF, Dhe-Paganon S, Donald LJ, Standing KG, Lee DC, Jia Z, Cyr TD.
    Journal: J Proteome Res; 2010 Jun 04; 9(6):3311-8. PubMed ID: 20405931.
    Abstract:
    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.
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