The use of chromium(III) to supercharge peptides by protonation at low basicity sites

The addition of chromium(III) nitrate to solutions of peptides with seven or more residues greatly increases the formation of doubly protonated peptides, [M + 2H](2+), by electrospray ionization. The test compound heptaalanine has only one highly basic site (the N-terminal amino group) and undergoes almost exclusive single protonation using standard solvents. When Cr(III) is added to the solution, abundant [M + 2H](2+) forms, which involves protonation of the peptide backbone or the C-terminus. Salts of Al(III), Mn(II), Fe(III), Fe(II), Cu(II), Zn (II), Rh(III), La(III), Ce(IV), and Eu(III) were also studied. Although several metal ions slightly enhance protonation, Cr(III) has by far the greatest ability to generate [M + 2H](2+). Cr(III) does not supercharge peptide methyl esters, which suggests that the mechanism involves interaction of Cr(III) with a carboxylic acid group. Other factors may include the high acidity of hexa-aquochromium(III) and the resistance of Cr(III) to reduction. Nitrate salts enhance protonation more than chloride salts and a molar ratio of 10:1 Cr(III):peptide produces the most intense [M + 2H](2+). Cr(III) also supercharges numerous other small peptides, including highly acidic species. For basic peptides, Cr(III) increases the charge state (2+ versus 1+) and causes the number of peptide molecules being protonated to double or triple. Chromium(III) does not supercharge the proteins cytochrome c and myoglobin. The ability of Cr(III) to enhance [M + 2H](2+) intensity may prove useful in tandem mass spectrometry because of the resulting overall increase in signal-to-noise ratio, the fact that [M + 2H](2+) generally dissociate more readily than [M + H](+), and the ability to produce [M + 2H](2+) precursors for electron-based dissociation techniques.