Analytical Chemistry of Proteins and Peptides

点击翻译按钮获取中文摘要

Advances in proteomics have been made possible by conceptual developments and technological breakthroughs in biological mass spectrometry (MS). The long-term objective of my Discovery Grant (DG) research program aims at a better understanding of the fundamental aspects of the science that underpins peptide and protein identification and characterization, as well as developing analytical technologies that are critical to this goal. Within the next DG period, we will explore the structure, formation, energetics and fragmentation chemistries of (1) protonated peptides ([M + H]+); (2) molecular radical cations of peptides (M+); and (3) metallated peptide complexes by means of a combination of experimental and theoretical approaches. Additionally, we aim at developing methods to detect, characterize, and quantify phosphorylated proteins and peptides. Experimental techniques will include: selective enrichment and liquid chromatography (LC), tandem MS with isotopic labeling, and infrared multiple photon dissociation (IRMPD) spectroscopy. To augment structural and mechanistic determinations, density functional theory (DFT) calculations will be employed. We will investigate the loss of water from [M + H]+; for oligopeptides comprising only alkyl residues, the source is the peptide bonds, which lose water competitively to give isomers of protonated imidazol-4-ones that can interconvert or dissociate independently. The effect of the residue side chain will be examined. We will also investigate the loss of water from M+, especially in peptides in which presence of the radical is apparently critical to water loss. Side-chain effects of residues will also be examined. We will fully explore the effects of lanthanides (Ln's), including Eu3+ and Ce3+, as the ionization agents in metallated peptides. Preliminary data indicate that Eu3+ and Ce3+ complexes exhibit very different chemistries, strongly suggesting different binding modes. We will undertake a new, major initiative in our DG research on phosphorylated peptides. We will determine by both theory and experiment the peptide structures which favour intramolecular phosphate migration (scrambling) under MS conditions. We will develop analytical technologies to enable selective enrichment of phosphopeptides for protein characterization as well as for determination of phosphoproteomes of cancers. Preliminary investigations have yielded a Zr4+-chelated adenosine triphosphate (ATP)-functionalized silica monolith that exhibited superior sensitivity and selectivity against -casein, a model phosphoprotein, which resulted in the observation of 19 out of a total of 20 known phosphosites and a detection limit of 1 fmol. We will develop the metal-ion-chelated ATP-functionalized silica monolith column in tandem with nanobore reverse-phase LC for determination of the phosphoproteomes of oral cancers and precancers that we have examined and for which biomarkers have been identified and commercialized.