title: Mass spectrometry following mild enzymatic digestion reveals phosphorylation of recombinant proteins in Escherichia coli through mechanisms involving direct nucleotide binding.
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.
authors.affiliation: Centre for Biologics Research, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa, Ontario, Canada.
authors.forename: Yi-Min
authors.initials: YM
authors.lastname: She
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Xu
authors.forename: Alexander F
authors.initials: AF
authors.lastname: Yakunin
authors.forename: Sirano
authors.initials: S
authors.lastname: Dhe-Paganon
authors.forename: Lynda J
authors.initials: LJ
authors.lastname: Donald
authors.forename: Kenneth G
authors.initials: KG
authors.lastname: Standing
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Lee
authors.forename: Zongchao
authors.initials: Z
authors.lastname: Jia
authors.forename: Terry D
authors.initials: TD
authors.lastname: Cyr
mesh_terms: D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000145:classification / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000145:classification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D006639:Histidine; D014357:Trypsin