Multiply charged negative ions by electrospray ionization of polypeptides and proteins

Abstract

Multiply deprotonated polypeptide and protein molecules, (M - nH)n-, produced from pH approximately 11 aqueous solutions, are analyzed by electrospray ionization-mass spectrometry (ESI-MS). Aqueous ammonium hydroxide solutions of the analyte are shown to be preferable to sodium hydroxide solutions for negative-ion ESI due to the production of multiply sodiated protein species from the latter system. Proteins with Mr to 66,000 and having up to 57 negative charges have been detected. Multiply charged negative ions can be produced from ESI of the highly acidic protein pepsin (Mr approximately 34,600) because of its relatively large number of acidic residues, 42. In contrast, the small number of basic amino acid residues for pepsin (4) does not allow formation of highly protonated species essential for positive-ion detection, for mass spectrometers of limited m/z range. Similarly, negative-ion ESI-MS is extended to large oligosaccharide analysis. Preliminary tandem mass spectrometry experiments of multiply charged polypeptide anions demonstrate the utility and potential of negative-ion ESI-MS for structural elucidation.

On the conformation‐dependent neutralization theory and charging of individual proteins and their non‐covalent complexes in the gas phase

“…42 -44 Earlier observations suggested correlation of the maximum charge state for the proteins electrosprayed from basic or acidic solutions with the respective number of acidic or basic residues in its sequence. 42,43 While the results obtained for the proteins within a molecular weight (MW) range of up to 40 kDa were qualitatively consistent with the proposed mechanism, significant deviations for higher molecular weight proteins were also reported. 42,43 Introduction of a proton transfer reactivity model, accounting for relative apparent gas-phase basicity of both multiply protonated protein ions and surrounding solvent molecules, enabled further progress to be made towards explaining experimental results obtained on a wider MW range.…”

“…42,43 While the results obtained for the proteins within a molecular weight (MW) range of up to 40 kDa were qualitatively consistent with the proposed mechanism, significant deviations for higher molecular weight proteins were also reported. 42,43 Introduction of a proton transfer reactivity model, accounting for relative apparent gas-phase basicity of both multiply protonated protein ions and surrounding solvent molecules, enabled further progress to be made towards explaining experimental results obtained on a wider MW range. 45 The success of this approach can be equally attributed to the realistic theoretical approximation of denatured proteins as elongated one-dimensional strings.…”

Statistical Characterization of Ion Trap Tandem Mass Spectra from Doubly Charged Tryptic Peptides

“…It has been noted in the literature that there is a strong preference for Pro to cleave at its N-terminal side, whether the product formed is the b ion or y ion. 30 Because b ions formed at most residues are accepted to have oxazolone structures (see Figure 1) [31][32][33] and because the b ion that would be formed at the C-terminal side of Pro would require a transition state involving formation of an unstable strained 5-5 bicyclic ring (see Figure 6), 6,8,34 cleavage typically occurs at the N-terminal side of Pro rather than the C-terminal side. This selective cleavage also provides information on the mechanism of the cleavage to form b and y ions.…”

“…The structure of Pro, for example, prevents the cleavage of the peptide bond C-terminal to the residue by hindering the attack of the N-terminal carbonyl 30 (see Figure 6). The extent to which each residue directionally enhances cleavage was measured by comparing the intensities of fragment ion peaks adjacent to each residue.…”

Vapor Treatment of Electrospray Droplets: Evidence for the Folding of Initially Denatured Proteins on the Sub-Millisecond Time-Scale

“…It has been noted, for example, that proteins present in solutions of low pH, in which significant denaturation is expected, give rise to higher average charge states in the positive polarity [4,9] than those prepared under conditions in which native structures are stable. Similarly, proteins ionized from a high pH solution tend to result in a higher average negative charge state in the negative polarity [10,11]. However, the CSD differences tend not to be as drastic as that noted in the positive polarity [12,13].…”