title: The utility of ETD mass spectrometry in proteomic analysis.
abstract: Mass spectrometry has played an integral role in the identification of proteins and their post-translational modifications (PTM). However, analysis of some PTMs, such as phosphorylation, sulfonation, and glycosylation, is difficult with collision-activated dissociation (CAD) since the modification is labile and preferentially lost over peptide backbone fragmentation, resulting in little to no peptide sequence information. The presence of multiple basic residues also makes peptides exceptionally difficult to sequence by conventional CAD mass spectrometry. Here we review the utility of electron transfer dissociation (ETD) mass spectrometry for sequence analysis of post-translationally modified and/or highly basic peptides. Phosphorylated, sulfonated, glycosylated, nitrosylated, disulfide bonded, methylated, acetylated, and highly basic peptides have been analyzed by CAD and ETD mass spectrometry. CAD fragmentation typically produced spectra showing limited peptide backbone fragmentation. However, when these peptides were fragmented using ETD, peptide backbone fragmentation produced a complete or almost complete series of ions and thus extensive peptide sequence information. In addition, labile PTMs remained intact. These examples illustrate the utility of ETD as an advantageous tool in proteomic research by readily identifying peptides resistant to analysis by CAD. A further benefit is the ability to analyze larger, non-tryptic peptides, allowing for the detection of multiple PTMs within the context of one another.
authors.affiliation: Department of Chemistry, University of Virginia, Charlottesville, VA 22904, USA.
authors.forename: Leann M
authors.initials: LM
authors.lastname: Mikesh
authors.forename: Beatrix
authors.initials: B
authors.lastname: Ueberheide
authors.forename: An
authors.initials: A
authors.lastname: Chi
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Coon
authors.forename: John E P
authors.initials: JE
authors.lastname: Syka
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Shabanowitz
authors.forename: Donald F
authors.initials: DF
authors.lastname: Hunt
mesh_terms: D000595:Amino Acid Sequence; D004220:Disulfides / Q000737:chemistry; D004579:Electron Transport; D004583:Electrons; D006031:Glycosylation; D006657:Histones / Q000737:chemistry; D007328:Insulin / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D020539:Sequence Analysis, Protein / Q000379:methods; D013451:Sulfonic Acids / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods; D025801:Ubiquitin / Q000737:chemistry
chemical_list: D004220:Disulfides; D006657:Histones; D007328:Insulin; D013451:Sulfonic Acids; D025801:Ubiquitin