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Proteomics. 2014 May;14(10):1152-7. doi: 10.1002/pmic.201300450. Epub 2014 Apr 17.

Artifacts to avoid while taking advantage of top-down mass spectrometry based detection of protein S-thiolation.

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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.


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.


Artifacts; Biomedicine; Cu/Zn-superoxide dismutase; Posttranslational modification; S-thiolation; Top down mass spectrometry

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