The Use of Chromium(III) to Supercharge Peptides by Protonation at Low Basicity Sites

Abstract

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. While 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 hexaaquochromium(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.

The Effects of Trivalent Lanthanide Cationization on the Electron Transfer Dissociation of Acidic Fibrinopeptide B and its Analogs

“…This is consistent with several studies suggesting that trivalent metal ions preferentially bind to acidic sites on peptides [50, 54-56, 58]. For example, in Figure 2 showing the ETD spectra of 2+, 3+, and 4+ precursors from fibrinopeptide B, only peptide fragments containing at least two acidic sites (i. e., glutamic acid or aspartic acid side chains and the C-terminal carboxylic acid group) are metallated.…”

“…We have previously found that trivalent chromium, Cr(III), readily cationizes acidic and neutral peptides [58, 68, 69] and that during CID these Cr(III)-peptide ions undergo extensive sequence-informative fragmentation [68]. Also, Cr(III) readily binds to acidic peptides in solution [10, 70, 71].…”

“…These experiments were unsuccessful because the ESI signal of iron(III)-cationized peptide was unstable and irreproducible. We have encountered this problem previously [58, 69] and attribute it to iron(III) reduction to iron(II) in solution (reduction potential, E 0 = +0.771 V [73]) during ESI. With an ESI source of similar design to our source (i.e., grounded needle with cations spraying into an electric field of negative polarity), Hoppilliard and coworkers [74] have also reported reduction of easily reduced metal cations.…”

“…Several studies demonstrate the ability of trivalent metal cation adduction to create quasi-molecular ions with increased charge states for small peptides in ESI [50, 54-58]. In the current work, the effects of lanthanide cationization on ETD of the model acidic peptide fibrinopeptide B and two of its analogs were studied.…”

The use of chromium(III) complexes to enhance peptide protonation by electrospray ionization mass spectrometry

“…Previously, we have shown that the addition of chromium(III) nitrate, [Cr(H 2 O) 6 ](NO 3 ) 3 ·3H 2 O, to peptide solutions undergoing ESI can enhance peptide protonation . Neutral and acidic peptides that normally only produce singly protonated molecules, [M + H] + , can generate abundant doubly protonated molecules, [M + 2H] 2+ , when trivalent chromium, Cr(III), is present in the solution being electrosprayed.…”