Artifacts to avoid while taking advantage of top-down mass spectrometry based detection of protein S-thiolation
Version of Record online: 17 APR 2014
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Special Issue: Top-down Proteomics
Volume 14, Issue 10, pages 1152–1157, May 2014
How to Cite
Auclair, J. R., Salisbury, J. P., Johnson, J. L., Petsko, G. A., Ringe, D., Bosco, D. A., Agar, N. Y. R., Santagata, S., Durham, H. D. and Agar, J. N. (2014), Artifacts to avoid while taking advantage of top-down mass spectrometry based detection of protein S-thiolation. Proteomics, 14: 1152–1157. doi: 10.1002/pmic.201300450
- Issue online: 10 MAY 2014
- Version of Record online: 17 APR 2014
- Accepted manuscript online: 13 MAR 2014 08:45AM EST
- Manuscript Accepted: 7 MAR 2014
- Manuscript Revised: 14 FEB 2014
- Manuscript Received: 10 OCT 2013
- National Institutes of Health. Grant Numbers: 1R01NS065263-01, 1R01NS067206-02
- ALS Therapy Alliance/CVS Pharmacy
- Fidelity Biosciences Research Initiative
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Supplemental Figure 1: Chemical Structures of S-cysteinylated (15963.38 Da) and S-thiolated (16149.44 Da) of SOD1 (15844.44 Da).
Supplemental Figure 2: Mechanisms of S-thiolation. Top: Thiolate-disulfide exchange can occur anaerobically with cysteine thiolate and any disulfide-containing small molecule. It is illustrated (top) for the reaction of cystine with SOD1, which yields Cys-bound-SOD1 (+119 Da) and free cysteine. Bottom: Disulfide bond formation starting from only sulfhydryl-containing compounds requires enzyme- or small molecule (e.g. oxygen)-mediated oxidation. It is illustrated (bottom) for the reaction of glutathione with SOD1.
Supplemental Figure 3: Thiol scavengers, iodoacetamide, iodoacetic acid, and MMTS, did not reverse cysteinylation of SOD1. Recombinant SOD1 was cysteinylated with 40-fold molar excess of L-cysteine. SOD1 was then incubated in the presence or absence of the thiol scavengers (iodoacetamide, iodoacetic acid, and/or MMTS) and homogenized using the same protocol that was used to homogenize tissue samples. After homogenization, the supernatants were incubated overnight (thiol scavengers still present) with end over end shaking to mimic purification of tissue homogenate with antibody conjugated beads. After overnight incubation, the samples were buffer exchanged into HPLC grade water and directly infused into a 9.4T FTMS. In all cases, SOD1 remained cysteinylated, thus the thiol scavengers used here did not appear to reverse SOD1 cysteinylation.
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