Ion mobility and mass spectrometry techniques are used to investigate the stabilities of different conformations of bradykinin (BK, Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg). At elevated solution temperatures, we observe a slow protonation reaction, i.e., [BK+2H]+H → [BK+3H], that is regulated by trans → cis isomerization of Arg-Pro, resulting in the Arg- cis-Pro- cis-Pro-Gly-Phe-Ser- cis-Pro-Phe-Arg (all- cis) configuration. Once formed, the all- cis [BK+3H] spontaneously cleaves the bond between Pro-Pro with perfect specificity, a bond that is biologically resistant to cleavage by any human enzyme. Temperature-dependent kinetics studies reveal details about the intrinsic peptide processing mechanism. We propose that nonenzymatic cleavage at Pro-Pro occurs through multiple intermediates and is regulated by trans → cis isomerization of Arg-Pro. From this mechanism, we can extract transition state thermochemistry: Δ G = 94.8 ± 0.2 kJ·mol, Δ H = 79.8 ± 0.2 kJ·mol, and Δ S = -50.4 ± 1.7 J·mol·K for the trans → cis protonation event; and, Δ G = 94.1 ± 9.2 kJ·mol, Δ H = 107.3 ± 9.2 kJ·mol, and Δ S = 44.4 ± 5.1 J·mol·K for bond cleavage. Biological resistance to the most favored intrinsic processing pathway prevents formation of Pro-Gly-Phe-Ser- cis-Pro-Phe-Arg that is approximately an order of magnitude more antigenic than BK.

Electrospray ionization (ESI) combined with ion mobility spectrometry (IMS) and mass spectrometry (MS) techniques is used to examine the Biginelli reaction in an ensemble of ions generated from droplets.