Artifacts to avoid while taking advantage of top-down mass spectrometry based detection of protein S-thiolation
| Autor: | Jeffrey N. Agar, Daryl A. Bosco, Joseph P. Salisbury, Sandro Santagata, Nathalie Y. R. Agar, Jared R. Auclair, Joshua L. Johnson, Gregory A. Petsko, Heather D. Durham, Dagmar Ringe |
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| Rok vydání: | 2013 |
| Předmět: |
Proteomics
Cystine chemistry.chemical_element Biochemistry Oxygen Mass Spectrometry Article Superoxide dismutase chemistry.chemical_compound Mice Animals Humans Sample preparation Cysteine Disulfides Molecular Biology Cerebral Cortex biology Superoxide Dismutase Proteins Small molecule chemistry biology.protein Artifacts Protein Processing Post-Translational Intracellular |
| Zdroj: | Proteomics. 14(10) |
| ISSN: | 1615-9861 |
| Popis: | Bottom-up mass spectrometry studies typically employ a reduction and alkylation step that eliminates a class of post-translational modification, 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. 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 post-mortem 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 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. |
| Databáze: | OpenAIRE |
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