High throughput screening of disulfide-containing proteins in a complex mixture

The formation of disulfide bonds between cysteine residues is crucial for the stabilization of native protein structures and, thus, determination of disulfide linkages is an important facet of protein structural characterization. Nonetheless, the identification of disulfide bond linkages remains a significant analytical challenge, particularly in large proteins with complex disulfide patterns. Herein, we have developed a new LC/MS strategy for rapid screening of disulfides in an intact protein mixture after a straightforward reduction step with tris(2-carboxyethyl)phosphine. LC/MS analysis of reduced and nonreduced protein mixtures quickly revealed disulfide-containing proteins owing to a 2 Da mass increase per disulfide reduction and, subsequently, the total number of disulfide bonds in the intact proteins could be determined. We have demonstrated the effectiveness of this method in a protein mixture composed of both disulfide-containing and disulfide-free proteins. Our method is simple (no need for proteolytic digestion, alkylation, or the removal of reducing agents prior to MS analysis), high throughput (fast on-line LC/MS analysis), and reliable (no S-S scrambling), underscoring its potential as a rapid disulfide screening method for proteomics applications.