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.
Jensen
P K
PK
Department of Chemistry and Ames Laboratory, USDOE, Iowa State University 50011, USA.
Harrata
A K
AK
Lee
C S
CS
D000478:Alkylation; D000818:Animals; D001709:Biotechnology; D002417:Cattle; D004220:Disulfides / Q000032:analysis / Q000737:chemistry*; D007461:Iodoacetates / Q000737:chemistry; D019807:Iodoacetic Acid; D007525:Isoelectric Focusing / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods; D008961:Models, Structural; D009862:Online Systems; D010084:Oxidation-Reduction; D010179:Pancreas / Q000201:enzymology; D011487:Protein Conformation; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D013438:Sulfhydryl Compounds / Q000737:chemistry
D004220:Disulfides; D007461:Iodoacetates; D013438:Sulfhydryl Compounds; D012259:Ribonuclease, Pancreatic; D019807:Iodoacetic Acid