Automatic determination of disulfide bridges in proteins

Precise determination of disulfide linkages between cysteine (Cys) residues in proteins is essential in the determination of protein structure. Therefore, a reliable automated method for the identification of disulfide bridges can serve as an important tool in the analysis of the tertiary structure of proteins of interest. Here, we describe the current and past methods used to identify disulfide bridges in proteins, with a focus on mass spectrometry (MS)-based methods and a particular emphasis on nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS)-based methods. We also show the development of an easy method based on the separation of disulfide-linked proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under denaturing and nonreducing conditions and selective in-gel digestion of proteins using reducing and nonreducing conditions, followed by analysis of the resulting peptide mixture by nanoACQUITY UPLC coupled to a quadrupole time-of-flight (QTOF) Micro mass spectrometer (nanoLC-MS/MS). Data-dependent analysis (DDA) nanoLC-MS/MS and information-dependent analysis (IDA) nanoLC-MS/MS were used for random and targeted identification of disulfide-linked peptides. Finally, an example of electrospray-MS (ESI-MS) and ESI-MS/MS-based determination of disulfide-linked peptides is shown.