Based on nickel-catalyzed cross-labeling where binding Partners become biotinylated. we have Studied molecular interactions with an N-terminally fused GGH-tag proinsulin C-peptide. Since C-peptide has been reported to influence phosphatase activity in intact cells, we employed this method to study possible binding of the peptide to protein tyrosine phosphatase 1B (PTP-1B). C-peptide Was found to interact with PTP-1B (and for control, also with antibodies to C-peptide), as did also the N- and C-terminal fragments of C-peptide which have sequence similarities with PIT-1B binding proteins. The labeling data combined with enzyme activity analysis indicate a functional interaction between acidic regions of C-peptide and specific sites of PTP-1B. Results highlight the importance of possible phosphatase/C-peptide roles in diabetes, and the Usefulness of the cross-labeling reaction also for acidic peptides like C-peptide. (C) 2009 Elsevier Inc. All rights reserved.