C-peptide structural and functional relationships studied by biosensor technology and mass spectrometry

Abstract

Proinsulin C-peptide has a number of biological activities and receives interest focusing on the therapeutic potential as a candidate for future co-replacement therapy with insulin in type 1 diabetes. Based on conservation results from comparisons of 22 mammalian proinsulin variants, analogs were constructed for studies of phosphorylation of mitogenactivated protein kinases (MAPKs) in Swiss 3T3 fibroblasts. The results show that phosphorylation of MAPKs is promoted by the presence of conserved glutamic acid residues at three positions of C-peptide and by a helical propensity in the Nterminal segment.

Degradation of C-peptide and its C-terminal pentapeptide was also studied. In serum, the bioactive pentapeptide was degraded by an aminopeptidase activity, while the full-length Cpeptide was endoproteolytically degraded. Serum proteins were removed using acetone precipitation, which made it possible to detect a novel N-terminal carbamate modification identified by tandem mass spectrometry. In kidney and placenta extracts, the degradation products were identified, showing major cleavages by an N-Leu-specific endoprotease, and minor aminopeptidase-like cleavages.

In attempts at purification of Cpeptide binding protein(s), novel microfluidic biosensor techniques were applied and developed. Utilizing surface plasmon resonance based biosensors, binding proteins were detected in human serum and in detergent-solubilized cellular and tissue material. Components were purified by interaction with biotinylated C-peptide attached to streptavidin-coated Biacore chips. The interaction was shown to be specific by lack of binding to scrambled C-peptide.

Proteins bound to the chip were eluted by micro-recovery techniques and were identified by fragment mass mappings and database searches. In all, ten proteins were identified in this manner from total extracts. Although several of the proteins are large and sticky, they show a pattern and demonstrate the power of affinity purification under simultaneous real-time monitoring of the binding.

We also studied a new biosensor chip, with a large gold surface (effective area 26 MM2), in affinity purification for enhanced protein recovery. In this manner, a 30-fold greater protein recovery than with conventional chips was demonstrated for an antiC-peptide antibody in a one-step purification from a protein mixture.

Finally, an immobilization technique was developed using an Attana biosensor instrument based on the quartz crystal microbalance technique. We demonstrate that charged molecules can be attracted to the chip surface for covalent attachment by application of a potential to the chip surface for electroimmobilization. The resulting chip was then used in biosensor capture of an anti-Cpeptide antibody, that was subsequently eluted using a two-bubble system for efficient recovery and mass spectrometric identification.

All three biosensor techniques studied are reproducible through repeated cycles and provide affinity purification of proteins under real-time monitoring of the binding and elution processes.