The objective of this research is to develop procedures for the analysis of acidic peptides by negative ion mass spectrometry. Many biological peptides and peptides used in disease treatment have numerous glutamic or aspartic acid residues; others contain acidic phosphate or sulfate groups. In the past fifteen years, advances in mass spectrometry have made it a method of choice for determining the molecular masses and sequences of peptides. Hundreds of reports have appeared on the tandem mass spectrometry (MS/MS) of positively-charged, protonated peptide ions. In contrast, relatively little work has focused on negatively- charged, deprotonated ions and the majority of these studies have dealt with singly charged ions containing four or fewer residues. However, acidic peptides often form negative ions more readily than positive ions. This project will utilize two advancing methodologies in mass spectrometry: electrospray ionization Fourier transform ion cyclotron resonance (ESI/FT-ICR) and matrix-assisted laser desorption ionization time-of-flight (MALDI/TOF). ESI/FT-ICR studies will involve low-energy collision-induced dissociation (CID) on multiply deprotonated ions, while MALDI/TOF work will employ post-source decay (PSD) on singly deprotonated ions. The major aim is to elucidate dissociation mechanisms. Fragmentations induced by various types of amino acid residues and by their sequences will be explored. Structural features under investigation include glutamic and aspartic acid residue, non- acidic residues such a proline and lysine, the C-terminal endgroup (-OH or NH2), highly acidic phosphate and sulfate groups, and interactions between acidic and basic residues. For multiply deprotonated ions generated by ESI, the number and locations of ionic charges (and thus the magnitude of Coulomb repulsion) must be considered. Molecular dynamics calculations will be performed to gain insight into Coulomb energy and conformations, which can affect dissociation. The impact of internal energy on fragmentation will be assessed by varying the parent ion excitation energy in the FT-ICR and, in the TOF, by augmenting PSD with collisions occurring after ions leave the source. Negative ion dissociation will be compared to that of the corresponding positive ions. In addition, structural factors that influence negative ion production will be investigated. To enhance deprotonated ion yields by MALDI, basic matrices will be developed for peptides.
