Establishing a Proteomics Workflow to Elucidate Disulfide Bonds While Minimizing Disulfide Scrambling
Hayward, John
Hayward, John
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Abstract
Disulfide bonds (DSBs) are covalent bonds that form between the sulfurs in two cysteine residues in a protein. This type of cross-linking maintains protein folding, providing stability to the protein structure and ensuring proper function. A common issue in identifying the cysteines involved in DSBs are the false positives reported as a result of DSB scrambling during sample preparation. Scrambled DSBs can be formed between non-crosslinked cysteines in already reduced, digested proteins, leading to the identification of non-native DSBs. To optimize the analytical method for both elucidating DSBs and minimizing DSB scrambling, two model proteins, ribonuclease A (RNase A) and bovine serum albumin (BSA). They were reduced under two conditions: high pH (ammonia bicarbonate buffered at pH 8.0) and low pH (Tris-HCL buffered at pH 6.5) and were alkylated with iodoacetamide or N-ethylmaleimide, respectively. Reduced/alkylated samples were digested using trypsin and/or chymotrypsin while maintaining pH 6.5 and 8.0. Resulting digests were analyzed using liquid chromatography-electrospray ionization tandem mass spectrometry (MS/MS) using collision-induced dissociation or higher-energy collisional dissociation on the Orbitrap-type mass spectrometers (Orbitrap Eclipse and Exploris 240). Protein coverages were compared in Proteome Discoverer (Thermo Fisher Scientific) to compare digestion efficiencies. Chromatographic elutions of the samples from different conditions were compared using the software Freestyle (Thermo Fisher Scientific) to pinpoint potential disulfide bonds formed under these conditions. Several software programs, including Protein Prospector (UCSF) and Spectrum Identification Machine, were used to search for possible DSBs and results were verified manually by de novo sequencing. Sites of disulfide bonds were compared to published results from Na Et al. and Rombouts Et al. work with RNase A and BSA, respectively.
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Date
1/1/2026
Student Status
Senior (Graduating in 2026)
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Poster
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Biochemistry
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College of Agriculture and Life Sciences
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Life Science