Quantification of Folded and Misfolded Proinsulin Forms Using Nonreducing SDS-PAGE and Proinsulin-Specific Immunoblotting

Abstract

We have observed that some proinsulin molecules in pancreatic islets and beta cell lines have incomplete or improper intramolecular disulfide bonds. These misfolded monomers can form intermolecular disulfide-linked complexes. Accurately quantifying the fraction of proinsulin molecules contained in these complexes is challenging. By proinsulin immunoblotting after nonreducing SDS-PAGE, the signal for disulfide-linked complexes can exceed the total proinsulin signal detected after reducing SDS-PAGE (i.e., overestimating the abundance of misfolded species due to antibody affinity differences). However, after modification of the SDS-PAGE and electrotransfer protocol, we have been able to more accurately estimate the fraction of proinsulin monomers folded to the native state, as well as misfolded proinsulin monomers and disulfide-linked complexes. Our improved technique offers the ability to obtain a more precise assessment of proinsulin misfolding under different environmental conditions in beta cells and normal islets and in diabetes. Key features • The protocol introduces a modification of a technique that enables clear separation and accurate quantification of native and non-native proinsulin monomers and aberrant disulfide-linked oligomers. • The protocol requires modifications to the standard SDS-PAGE and electrotransfer protocol to address quantitation inaccuracies that result from variations in antibody affinity. • Side-by-side comparison demonstrates that the standard immunoblotting method underestimates proinsulin monomers and overestimates the abundance of proinsulin disulfide-linked complexes. • This method is applicable to the study of recombinant proinsulin in heterologous cells, pancreatic β-cell lines, rodent or human pancreatic islets, and human iPSCs.

Keywords: Beta cells; Disulfide bonds; Proinsulin misfolding; Quantitation.