Gas-Phase Chemistry of Biological and Metal-Containing Ions

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Advances in proteomics have been made possible by conceptual developments and technological breakthroughs in biological mass spectrometry (MS). The long-term objective of our Discovery research program will continue to aim at better understanding fundamental aspects of the science that underpins peptide and protein identification and characterization. In the shorter term, and within the duration of this funding cycle, our objectives are to explore the structure, formation, energetics and fragmentation chemistry of (1) protonated peptides ([M + H]+) and their key fragments; (2) molecular radical cations of peptides (M•+); and (3) metalated peptide complexes. These will be tackled using a combination of experimental and theoretical approaches. Experimental techniques will include: (i) tandem MS with isotopic labeling with 13C, 2H, 18O and 15N; (ii) threshold collision-induced dissociation (CID) on a modified MS instrument fitted with a ring ion guide as q2; and (iii) IRMPD spectroscopy performed in the FELIX facility. We propose herein to continually improve our understanding of the structures and dissociation chemistry of protonated peptides and their key fragments. We will start with selected a(n) product ions and their next-generation fragments formed after losing a small neutral product, such as ammonia giving a(n)*, and water giving a(n)0 ions. We will examine the effects of side-chain functional groups on the relative energetics and stability of a(2) isomers, especially those of a newly discovered imine-amide structure (L2) and a cyclic structure (C1), by performing a systematic study including basic and acidic residues. Relatively little is known about the structures of a(n)* and a(n)0 ions. Our preliminary results strongly suggest that the a(3)* and a(4)* ions of pentaalanine (A5) have unconventional structures in that they have imine-oxazolone structures. Surprisingly, the a(4)* ion of pentaglycine (G5) is not an imine-oxazolone, but a protonated imidazolone, a new class of products first proposed by us for dehydrated protonated G4, a nominal b4 ion. In addition, the IRMPD spectrum of the a(4)* ion of G5 closely resembles that of the a(4)0, thereby suggesting both structures are protonated imidazolones. We will fully explore and determine the mechanisms and energetics leading to the different isomeric products. We will continue improving our understanding of factors that determine structural stability in peptide radical ions and their fragmentation mechanisms. CID of M•+ from oligopeptides of glycine containing a single tyrosine (Y) or tryptophan (W) residue in a non-terminal position typically shows most-prominent products that originate from a proton-bound ‘dimer’ formed after cleavage of the N–C(alpha) bond of Y or W. Surprisingly, preliminary data on the CID of oligoalanines containing a single Y residue reveal additional and puzzling fragmentation channels. We plan to fully investigate these fascinating fragmentation channels by ‘moving’ Y along the oligoalanine framework and having Y as the third, fourth and subsequent residues. In addition, we will also examine CID results of the M•+ from oligoalanines containing a single W residue. We plan to continue examining multi-stage MS of metalated peptide complexes to generate unusual and fascinating peptide ions, including dispositive protonated a3 and a2 ions which have high charge-density due to their small size. We attribute the effectiveness of LaIII in generating these ions to its high charge and its propensity to delocalize its charge onto the ligand, as well as the high affinity of La for O (thereby facilitating the cleavage of COO-). We plan to extend the line of study to using Ce•3+ and other lanthanide ions as the metalation center.