At high pH and in the presence of dissolved CO 2 , the N-terminus and ε-amino groups of amino acids, peptides, and proteins can form carbamino adducts with CO 2 , R-NH 2 + CO 2 ↔ R-NHCOO − + H + . We report the first study of carbamino group formation by electrospray ionization (ESI) mass spectrometry (MS). Angiotensin II, bradykinin, substance P, and insulin have been studied. A careful optimization of the instrumental parameters was necessary to allow the transfer of the fragile adducts into vacuum for mass analysis. Particularly, dissociation of the adducts in the ion sampling process and pH changes in ESI must be minimized. With these precautions, levels of carbamino group formation of angiotensin II and bradykinin determined from mass spectra agree with those expected to be in solution, calculated from literature equilibrium constants. Thus, ESI MS can quantitatively measure ratios of carbamino adduct to total peptide concentration in solution. Values of equilibrium constants for carbamino group formation with substance P (pK c = 4.77 ± 0.18) and insulin (pK c = 4.99 ± 0.05) are reported for the first time.