title: Improved identification and relative quantification of sites of peptide and protein oxidation for hydroxyl radical footprinting.
abstract: Protein oxidation is typically associated with oxidative stress and aging and affects protein function in normal and pathological processes. Additionally, deliberate oxidative labeling is used to probe protein structure and protein-ligand interactions in hydroxyl radical protein footprinting (HRPF). Oxidation often occurs at multiple sites, leading to mixtures of oxidation isomers that differ only by the site of modification. We utilized sets of synthetic, isomeric "oxidized" peptides to test and compare the ability of electron-transfer dissociation (ETD) and collision-induced dissociation (CID), as well as nano-ultra high performance liquid chromatography (nanoUPLC) separation, to quantitate oxidation isomers with one oxidation at multiple adjacent sites in mixtures of peptides. Tandem mass spectrometry by ETD generates fragment ion ratios that accurately report on relative oxidative modification extent on specific sites, regardless of the charge state of the precursor ion. Conversely, CID was found to generate quantitative MS/MS product ions only at the higher precursor charge state. Oxidized isomers having multiple sites of oxidation in each of two peptide sequences in HRPF product of protein Robo-1 Ig1-2, a protein involved in nervous system axon guidance, were also identified and the oxidation extent at each residue was quantified by ETD without prior liquid chromatography (LC) separation. ETD has proven to be a reliable technique for simultaneous identification and relative quantification of a variety of functionally different oxidation isomers, and is a valuable tool for the study of oxidative stress, as well as for improving spatial resolution for HRPF studies.
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia, Athens, GA, 30602, USA.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Li
authors.forename: Zixuan
authors.initials: Z
authors.lastname: Li
authors.forename: Boer
authors.initials: B
authors.lastname: Xie
authors.forename: Joshua S
authors.initials: JS
authors.lastname: Sharp
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004583:Electrons; D006801:Humans; D017665:Hydroxyl Radical; D009419:Nerve Tissue Proteins / Q000737:chemistry; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification; D023302:Protein Footprinting / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D011971:Receptors, Immunologic / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods; D000097905:Roundabout Proteins
chemical_list: D017665:Hydroxyl Radical; D009419:Nerve Tissue Proteins; D010455:Peptides; D011506:Proteins; D011971:Receptors, Immunologic; D000097905:Roundabout Proteins