Enhanced Protonation of Peptides Using Trivalent Chromium in Mass Spectrometry

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Ionization of peptides is an important step in mass spectrometry (MS)-based bottom-up proteomics. Electrospray ionization (ESI) is a common method of converting peptide ions into the gas phase. ESI is known to produce multiply protonated ions, [M + nH]n+. The addition of trivalent chromium, Cr(III), to peptide solutions undergoing ESI was discovered to increase the protonation of peptides. This dissertation contains fundamental studies of peptide ionization using trivalent chromium, Cr(III), as an additive.Experimental studies were completed to deduce the mechanism of Cr(III) enhanced protonation in ESI. Cr(III) enhances the protonation of model peptides with an acidic residue at the C-terminus to a greater extent than when the acidic residue is further away. The protonation of model peptide amides containing no carboxyl groups at the C-terminus or sidechain are enhanced by the addition of Cr(III). This indicates that carboxyl and amide groups are involved in the mechanism. Enhanced protonation with Cr(III) was ineffective with phosphorylated peptides and contributed to a pH effect in proteins.A survey of twenty-seven biological peptides was completed to gauge the analytical utility of Cr(III) in MS-based proteomics. Not all peptides underwent enhanced protonation by ESI upon addition of Cr(III), but Cr(III) did enhance the protonation of eleven peptides by adding an additional proton or increasing the signal intensity. Compared to model peptides, the interactions between sidechains of biological peptides are of higher complexity and can prevent the binding and/or dissociation of Cr(III) from the peptide.Different methods of delivering Cr(III) into the ESI source region were attempted to incorporate Cr(III) in MS-based proteomic workflows. Post-column addition of Cr(III) is possible using a tee union to introduce the Cr(III) solution or by doping the nebulizing gas with Cr(III). This signifies that Cr(III) does not have to be added to peptide solutions but can be introduced with the nebulizing gas during desolvation or as a separate solution into the ESI source bypassing chromatography. Dissociation of protonated ions formed by Cr(III) were also studied. The electron transfer dissociation (ETD) products of precursor ions generated upon addition of Cr(III) did not differ from those of precursor ions generated using 1% acetic acid. Higher charge states (n) generate better sequence coverage than [M + 2H]2+. Certain residues (i.e., proline, basic, and acid residues) direct fragmentation and the type of product ions produced in ETD. This residue effect is more prevalent with [M + 2H]2+ and subsides with higher charge states. Matrix-assisted laser desorption ionization (MALDI) is another method of ionizing peptides. Singly protonated ions, [M +H]+, are generally formed in MALDI. Additional protons are not expected to be added with Cr(III), but rather an increase in the intensity of [M + H]+. Although some promising results were obtained, the effect of Cr(III) as an additive in MALDI analysis of peptides was inconclusive due to poor reproducibility, which is common in MALDI.

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