title: Characterization of S-thiolation on secreted proteins from E. coli by mass spectrometry.
abstract: S-thiolation is a reversible post-translational modification in which thiol metabolites of low molecular masses are linked to protein sulfhydryl groups through disulfide bonds. This modification is commonly observed in recombinant proteins secreted from E. coli cells. Since it can alter protein functions and introduce molecular heterogeneity, S-thiolation is undesirable for recombinant protein production. To date, few published studies have characterized thiol modifiers or investigated the mechanism of S-thiolation in recombinant proteins. In this work, reversed-phase liquid chromatography and mass spectrometry were used to characterize four of the most abundant thiol modifiers on recombinant proteins secreted from E. coli BL21 (DE3) strain. These thiol modifiers have been identified as glutathione, 4-phosphopantetheine, gluconoylated glutathione, and dephosphorylated coenzyme A. S-thiolation by these thiol modifiers increases protein mass by 305, 356, 483, and 685 Da, respectively. These specific mass increases can be used as markers for identifying S-thiolation in recombinant proteins.
authors.affiliation: Department of Protein Therapeutics Development, Bristol-Myers Squibb Research and Development, P.O. Box 5400, Princeton, NJ 08543, USA.
authors.forename: Peiran
authors.initials: P
authors.lastname: Liu
authors.forename: Malloree A
authors.initials: MA
authors.lastname: Tarnowski
authors.forename: Brian W
authors.initials: BW
authors.lastname: O'Mara
authors.forename: Wei
authors.initials: W
authors.lastname: Wu
authors.forename: Haiying
authors.initials: H
authors.lastname: Zhang
authors.forename: James K
authors.initials: JK
authors.lastname: Tamura
authors.forename: Michael S
authors.initials: MS
authors.lastname: Ackerman
authors.forename: Li
authors.initials: L
authors.lastname: Tao
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Grace
authors.forename: Reb J
authors.initials: RJ
authors.lastname: Russell
mesh_terms: D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D008970:Molecular Weight; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds