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TitleArtifacts to avoid while taking advantage of top-down mass spectrometry based detection of protein S-thiolation.
Publication TypeJournal Article
Year of Publication2014
AuthorsAuclair JR, Salisbury JP, Johnson JL, Petsko GA, Ringe D, Bosco DA, Agar NYR, Santagata S, Durham HD, Agar JN
JournalProteomics
Volume14
Issue10
Pagination1152-7
Date Published2014 May
ISSN1615-9861
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.

DOI10.1002/pmic.201300450
Alternate JournalProteomics
PubMed ID24634066
Grant List1R01NS065263-01 / NS / NINDS NIH HHS / United States
1R01NS067206-02 / NS / NINDS NIH HHS / United States
R01 NS065263 / NS / NINDS NIH HHS / United States