Identification of disulfide bond demands careful planning of enzymatic digestion stages

Disulfide bonds promote folding, and stabilize tertiary structure of a protein. Correct folding is necessary to achieve the three-dimensional conformation required for the catalytic activity of enzymes that regulates various biological pathways in a cell.  

Disulfide bonds can be identified by LC-MS/MS analysis. However, too short peptides can result in false identifications.  

Selection of correct digestion enzymes and incubation time to generate peptides of desirable length can successfully identify disulfide bonds in a protein. Na et al., have used a combination of Asp-N/C and trypsin for MS/MS-based analysis of disulfide bonds in RNase A. They also regulated the digestion time to deliberately introduce missed trypsin cleavage and produce cross-linked peptides of appropriate lengths for LC-MS/MS.

RNase A contains four disulfide bonds in its native state. Figure (1) shows that only Asp-N/C + LysC combination generates simple single inter-disulfide-linked peptides with a length suitable for analysis by MS/MS. Based on digestion yield and specificity, Na et al., instead used Asp-N/C + trypsin combination. A missed cleavage was introduced to increase the length of peptide that includes Cys84 (see panel (d) in the figure).

DBond software was used for the analysis of disulfide bonds in the protein.

Thus, careful selection of digestion enzymes and missed cleavage reactions can lead to confident identification of disulfide bond in a protein.

Reference:

Na S, Paek E, Choi J-C, Kim D, Lee S-J, and Kwon J (2015) Characterization of disulfide bonds by planned digestion and tandem mass spectrometry. Molecular BioSystems