The amino acid sequences of peptides affect their biological activity and, consequently, their utility in the diagnosis and treatment of human health issues. The overall objective of this research is to develop procedures for analyzing and sequencing acidic and neutral peptides by mass spectrometry (MS) using electron-induced dissociation techniques. The focus will be on peptides that are difficult to sequence by current mass spectrometry procedures. The first specific aim involves the use of trivalent metal salts to enhance the protonation of peptides by electrospray ionization (ESI). This will result in doubly protonated ions for peptides that normally only singly protonate. This important because the most common electron-induced fragmentation processes, electron transfer dissociation (ETD) and electron capture dissociation (ECD), require that the precursor ion be a multiply charged cation. Trivalent salts of chromium, Cr(III), have been shown to add a second proton to small peptides that normally singly protonate because they contain only one basic site. Other trivalent metals, such as Fe(III), Rh(III), Al(III), and Eu(III), will also be studied to determine if they enhance protonation for small peptides. The mechanism of this effect with is probed. The salt that produces optimum protonation will be evaluated with mixtures of peptides to determine if peptide ion suppression occurs and also to investigate the impact of the salt on chromatographic separations. The second specific aim is to explore ETD and ECD fragmentation pathways that result from multiply protonated peptide ions generated with trivalent metal ions. The development of a trivalent metal reagent should allow analysis of peptide types that has never been studied before by ETD or ECD;for example, it will now be possible to study acidic peptides and neutral peptides with alkyl side chains. Dissociation mechanisms and fragment ion structures with be probed using multi-stage mass spectrometry, including combinations involving collision-induced dissociation (CID) as a second stage. In addition to enhancing protonation, ESI on mixtures of metal salts and peptides often results in formation of metal-cationized peptide ions. For trivalent metals, these complex ions may have added or removed protons from the peptide, resulting in complexes with charges of 2+, 3+, or 4+. The ability of these complexes to undergo ETD or ECD and yield sequence information for a variety of acidic and neutral peptides will be explored. The lanthanide series of trivalent ions wil be studied in detail. The third member of the series, praseodymium (Pr) with atomic number 59, appears particularly suitable for peptide sequencing. Factors to consider in characterizing the use of trivalent metal ion complexes to sequence peptides include metal electron configuration and ionic radius, the location of the metal ion and the extent of protonation or deprotonation in the precursor and product ions, and the types, number, and locations of amino acid residues in the peptide sequence. Density functional theory (DFT) calculations will be employed to gain insight into the structures and energetics involved in these processes.
The sequence of a peptide influences its biological function and its ability to cause or treat human health issues. Mass spectrometry (MS) is commonly employed to analyze peptides, but is not always successful in obtaining their sequences. The proposed work will explore the ability of electron-induced MS dissociation techniques to sequence peptides that have been multiply charged using trivalent metal ions.