Electrospray ionization (ESI) on mixtures of acidic fibrinopeptide B and two peptide analogs with trivalent lanthanide salts generates [M + Met + H]4+, [M + Met]3+, and [M + Met –H]2+, where M = peptide and Met = metal (except radioactive promethium). These ions undergo extensive and highly efficient electron transfer dissociation (ETD) to form metallated and non-metallated c- and z-ions. All metal adducted product ions contain at least two acidic sites, which suggest attachment of the lanthanide cation at the side chains of one or more acidic residues. The three peptides undergo similar fragmentation. ETD on [M + Met + H]4+ leads to cleavage at every residue; the presence of both a metal ion and an extra proton is very effective in promoting sequence-informative fragmentation. Backbone dissociation of [M + Met]3+ is also extensive, although cleavage does not always occur between adjacent glutamic acid residues. For [M + Met – H ]2+, a more limited range of product ions form. All lanthanide metal peptide complexes display similar fragmentation except for europium (Eu). ETD on [M + Eu – H]2+ and [M + Eu]3+ yields a limited amount of peptide backbone cleavage; however, [M + Eu + H]4+ dissociates extensively with cleavage at every residue. With the exception of the results for Eu(III), metallated peptide ion formation by ESI, ETD fragmentation efficiencies, and product ion formation are unaffected by the identity of the lanthanide cation. Adduction with trivalent lanthanide metal ions is a promising tool for sequence analysis of acidic peptides by ETD.

Matrix-assisted laser desorption/ionization (MALDI) in-source decay was studied in the negative ion mode on deprotonated peptides to determine its usefulness for obtaining extensive sequence information for acidic peptides. Eight biological acidic peptides, ranging in size from 11 to 33 residues, were studied by negative ion mode ISD (nISD). The matrices 2,5-dihydroxybenzoic acid, 2-aminobenzoic acid, 2-aminobenzamide, 1,5-diaminonaphthalene, 5-amino-1-naphthol, 3-aminoquinoline, and 9-aminoacridine were used with each peptide. Optimal fragmentation was produced with 1,5-diaminonphthalene (DAN), and extensive sequence informative fragmentation was observed for every peptide except hirudin(54–65). Cleavage at the N–Cα bond of the peptide backbone, producing c′ and z′ ions, was dominant for all peptides. Cleavage of the N–Cα bond N-terminal to proline residues was not observed. The formation of c and z ions is also found in electron transfer dissociation (ETD), electron capture dissociation (ECD), and positive ion mode ISD, which are considered to be radical-driven techniques. Oxidized insulin chain A, which has four highly acidic oxidized cysteine residues, had less extensive fragmentation. This peptide also exhibited the only charged localized fragmentation, with more pronounced product ion formation adjacent to the highly acidic residues. In addition, spectra were obtained by positive ion mode ISD for each protonated peptide; more sequence informative fragmentation was observed via nISD for all peptides. Three of the peptides studied had no product ion formation in ISD, but extensive sequence informative fragmentation was found in their nISD spectra. The results of this study indicate that nISD can be used to readily obtain sequence information for acidic peptides.

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.