Monitoring protein refolding induced by disulfide formation using capillary isoelectric focusing-electrospray ionization mass spectrometry.

Rapid growth in the biotechnology industry has led to a dramatic increase in attention to the protein folding problem. Understanding protein-folding pathways is essential to the production of biopharmaceuticals since commercial production of recombinant proteins often requires a protein-refolding process for recovery of high yields. Protein folding coupled to the formation of disulfide bonds presents one of the simplest approaches to studying folding intermediates. On-line capillary isoelectric focusing-electrospray ionization mass spectrometry (CIEF-ESIMS) is demonstrated for kinetic studies of disulfide bond-induced protein refolding. Refolding intermediates of bovine pancreatic ribonuclease A, a model system for this study, are blocked at different stages by alkylating free thiols with iodoacetate. The alkylation reaction results in the introduction of charge (-1) and mass (59) differences for each alkylation site, providing the means for predictable separation and direct identification of refolding intermediates using CIEF-ESIMS. Besides the observation of refolding intermediates containing different numbers of disulfide bonds and even mixed disulfides, the two-dimensional resolving power of CIEF-ESIMS allows the determination of conformational heterogeneity among groups of refolding intermediates.