Ion mobility and mass spectrometry techniques are used to investigate the stabilities of different conformations of bradykinin (BK, Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)). At elevated solution temperatures, we observe a slow protonation reaction, i.e., [BK+2H](2+)+H+ -> [BK+3H](3+), that is regulated by trans -> cis isomerization of Arg(1)-Pro(2), resulting in the Arg(1)-cis-Pro(2)-cis-Pro(3)-Gly(4)-Phe(5)-Ser(6)-cis-Pro(7)-Phe(8)-Arg(9) (all-cis) configuration. Once formed, the all-cis [BK+3H](3+) spontaneously cleaves the bond between Pro(2)-Pro(3) 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(2)-Pro(3) occurs through multiple intermediates and is regulated by trans -> cis isomerization of Arg(1)-Pro(2). From this mechanism, we can extract transition state thermochemistry: Delta G(double dagger) = 94.8 +/- 0.2 kJ.mol(-1), Delta H-double dagger = 79.8 +/- 0.2 kJ.mol(-1), and Delta S-double dagger = -50.4 +/- 1.7 J.mol(-1).K-1 for the trans -> cis protonation event; and, Delta G(double dagger) = 94.1 +/- 9.2 kJ.mol(-1), Delta H-double dagger = 107.3 +/- 9.2 kJ.mol(-1), and Delta S-double dagger = 44.4 +/- 5.1 J.mol(-1).K-1 for bond cleavage. Biological resistance to the most favored intrinsic processing pathway prevents formation of Pro(3)-Gly(4)-Phe(5)-Ser(6)-cis-Pro(7)-Phe(8)-Arg(9) that is approximately an order of magnitude more antigenic than BK.