title: Artifacts to avoid while taking advantage of top-down mass spectrometry based detection of protein S-thiolation.
abstract: Bottom-up MS studies typically employ a reduction and alkylation step that eliminates a class of PTM, S-thiolation. Given that molecular oxygen can mediate S-thiolation from reduced thiols, which are abundant in the reducing intracellular milieu, we investigated the possibility that some S-thiolation modifications are artifacts of protein preparation. Cu/Zn-superoxide dismutase (SOD1) was chosen for this case study as it has a reactive surface cysteine residue, which is readily cysteinylated in vitro. The ability of oxygen to generate S-thiolation artifacts was tested by comparing purification of SOD1 from postmortem human cerebral cortex under aerobic and anaerobic conditions. S-thiolation was ∼50% higher in aerobically processed preparations, consistent with oxygen-dependent artifactual S-thiolation. The ability of endogenous small molecule disulfides (e.g. cystine) to participate in artifactual S-thiolation was tested by blocking reactive protein cysteine residues during anaerobic homogenization. A 50-fold reduction in S-thiolation occurred indicating that the majority of S-thiolation observed aerobically was artifact. Tissue-specific artifacts were explored by comparing brain- and blood-derived protein, with remarkably more artifacts observed in brain-derived SOD1. Given the potential for such artifacts, rules of thumb for sample preparation are provided. This study demonstrates that without taking extraordinary precaution, artifactual S-thiolation of highly reactive, surface-exposed, cysteine residues can result.
authors.affiliation: Department of Chemistry and Chemical Biology, Barnett Institute, Northeastern University, Boston, MA, USA; Department of Pharmaceutical Sciences, Northeastern University, Boston, MA, USA; Department of Biochemistry, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA, USA; Department of Chemistry, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA, USA.
authors.forename: Jared R
authors.initials: JR
authors.lastname: Auclair
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Salisbury
authors.forename: Joshua L
authors.initials: JL
authors.lastname: Johnson
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Petsko
authors.forename: Dagmar
authors.initials: D
authors.lastname: Ringe
authors.forename: Daryl A
authors.initials: DA
authors.lastname: Bosco
authors.forename: Nathalie Y R
authors.initials: NY
authors.lastname: Agar
authors.forename: Sandro
authors.initials: S
authors.lastname: Santagata
authors.forename: Heather D
authors.initials: HD
authors.lastname: Durham
authors.forename: Jeffrey N
authors.initials: JN
authors.lastname: Agar
mesh_terms: D000818:Animals; D016477:Artifacts; D002540:Cerebral Cortex / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D013482:Superoxide Dismutase / Q000737:chemistry
chemical_list: D004220:Disulfides; D011506:Proteins; D013482:Superoxide Dismutase; D003545:Cysteine
keywords: Artifacts; Biomedicine; Cu/Zn-superoxide dismutase; Posttranslational modification; S-thiolation; Top down mass spectrometry