Localization of Disulfide Bonds in Ribonuclease Using Low pH Trypsin and LC-ESI-QTOF-MS

We evaluated a method to localize disulfide bonds in bovine pancreatic ribonuclease (RNase) by applying low pH trypsin protein digestion with a bottom-up LC-ESI-QTOF-MS approach. The goal was to minimize disulfide bond scrambling during sample preparation. By using N-ethylmaleimide (NEM) for alkylation of free cysteines, we achieved 92% sequence coverage and successfully identified the native disulfide bonds between specific cysteine residues. However, scrambled disulfide bonds were also observed. Our results indicate that while this low pH digestion method helps preserve native disulfide bonds, further refinement is needed to fully prevent disulfide bond rearrangement during sample preparation.