Optimization of instrument and sample preparation factors for enhanced protonation with and without Cr(III) is necessary to allow maximum formation of [M + 2H] . Proteomics researchers should find these procedures to be of use for increasing multiply protonated signal intensity even in the absence of Cr(III). Copyright © 2017 John Wiley & Sons, Ltd.

The addition of trivalent chromium, Cr(III), reagents to peptide solutions can increase the intensity of doubly protonated peptides, [M + 2H]2+, through electrospray ionization (ESI). Three model heptapeptides were studied: neutral (AAAAAAA), acidic (AAEEEAA), and basic (AAAKAAA). The neutral and acidic peptides form almost no 2+ ions in the absence of Cr(III). Twenty Cr(III) complexes were used as potential enhanced protonation reagents, including 11 complexes with nonlabile ligands and nine with labile ligands. The complexes that provide the most abundant [M + 2H]2+, the greatest [M + 2H]2+ to [M + H]+ ratio, and the cleanest mass spectra are [Cr(H2O)6](NO3)3·3H2O and [Cr(THF)3]Cl3. Anions in Cr(III) reagents can also affect the intensity of [M + 2H]2+ and the [M + 2H]2+ to [M + H]+ ratio through cation‐anion interactions. The influence of anions on the extent of peptide protonation follows the trend ClO4− ˃ SO42− ˃ Br− ˃ Cl− ˃ F− ≈ NO3−. Solvent effects and complexes with varying number of water ligands were investigated to study the importance of water in enhanced protonation. Aqueous solvent systems and Cr(III) complexes that have at least one bound water ligand in solution must be used for successful increase in the intensity of [M + 2H]2+, which indicates that water is involved in the mechanism of Cr(III)‐induced enhanced protonation. The ESI source design is also important because no enhanced protonation was observed using a Z‐spray source. The current results suggest that this Cr(III)‐induced effect occurs during the ESI desolvation process.

Addition of trivalent chromium, Cr­(III), to solutions undergoing electrospray ionization (ESI) enhances protonation and leads to formation of [M + 2H]2+ for peptides that normally produce [M + H]+. This effect is explored using electronic structure calculations at the density functional theory (DFT) level to predict the energetics of various species that are potentially important to the mechanism. Gas- and solution-phase reaction free energies for glycine and its anion reacting with [Cr­(III)­(H2O)6]3+ and for dehydration of these species have been predicted, where glycine is used as a simple model for a peptide. For comparison, calculations were also performed with Fe­(III), Al­(III), Sc­(III), Y­(III), and La­(III). Removal of water from these complexes, as would occur during the ESI desolvation process, results in species that are highly acidic. The calculated pK a of Cr­(III) with a single solvation shell is −10.8, making [Cr­(III)­(H2O)6]3+ a superacid that is more acidic than sulfuric acid (pK a = −8.8). Binding to glycine requires removal of two aqua ligands, which gives [Cr­(III)­(H2O)4]3+ that has an extremely acidic pK a of −28.8. Removal of additional water further enhances acidity, reaching a pK a of −84.7 for [Cr­(III)­(H2O)]3+. A mechanism for enhanced protonation is proposed that incorporates computational and experiment results, as well as information on the known chemistry of Cr­(III), which is substitutionally inert. The initial step involves binding of [Cr­(III)­(H2O)4]3+ to the deprotonated C-terminus of a peptide. As the drying process during ESI strips water from the complex, the resulting superacid transfers protons to the bound peptide, eventually leading to formation of [M + 2H]2+.