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Filename: controllers/Detail.php
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Filename: controllers/Detail.php
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Filename: models/Detail_model.php
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Function: insertAPISummary
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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.
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http://dx.doi.org/10.1002/pmic.201300450 | DOI Listing |
Redox Biol
June 2018
Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton; NIHR Southampton Biomedical Research Centre, University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton, UK. Electronic address:
Several diseases are associated with perturbations in redox signaling and aberrant hydrogen sulfide metabolism, and numerous analytical methods exist for the measurement of the sulfur-containing species affected. However, uncertainty remains about their concentrations and speciation in cells/biofluids, perhaps in part due to differences in sample processing and detection principles. Using ultrahigh-performance liquid chromatography in combination with electrospray-ionization tandem mass spectrometry we here outline a specific and sensitive platform for the simultaneous measurement of 12 analytes, including total and free thiols, their disulfides and sulfide in complex biological matrices such as blood, saliva and urine.
View Article and Find Full Text PDFProteomics
May 2014
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.
View Article and Find Full Text PDFBiochim Biophys Acta
January 1985
Protein mixed-disulfides in cultured rat heart cells were analyzed by gel electrophoresis under conditions that eliminated artifactual formation of these protein derivatives. Protein S-thiolation (protein mixed-disulfide formation) was detectable under normal culture conditions. Diamide oxidized intracellular glutathione in these cells and produced extensive protein S-thiolation.
View Article and Find Full Text PDFEnter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!