title: A modified database search strategy leads to improved identification of in vitro brominated peptides spiked into a complex proteomic sample.
abstract: Inflammation leads to activation of immune cells, resulting in production of hypobromous acid. Few investigations have been performed on protein bromination on a proteomic scale, even though bromination is a relatively abundant protein modification in endogenously brominated proteomes. Such studies have been hampered by the lack of an optimized database search strategy. In order to address this issue, we performed nano-LC-MS/MS analysis of an in vitro generated, trypsin-digested brominated human serum albumin standard, spiked into a complex trypsin-digested proteomic background, in an LTQ-Orbitrap instrument. We found that brominated peptides spiked in at a 1-10% ratio (mass:mass) were easily identified by manual inspection when higher-energy collisional dissociation (HCD) and collision induced dissociation (CID) were employed as the dissociation mode; however, confident assignment of brominated peptides from protein database searches required a novel approach. By addition of a custom modification, corresponding to the substitution of a single bromine with 81Br rather than 79Br for dibromotyrosine (79Br81BrY), the number of validated assignments for peptides containing dibromotyrosine increased significantly when analyzing both high resolution and low resolution MS/MS data. This new approach will facilitate the identification of proteins derived from endogenously brominated proteomes, providing further knowledge about the role of protein bromination in various pathological states.
authors.affiliation: University of Texas Medical Branch, Department of Pharmacology and Toxicology, 301 University Boulevard, Galveston, Texas 77555-0617, United States.
authors.forename: Huiling
authors.initials: H
authors.lastname: Liu
authors.forename: Cheryl F
authors.initials: CF
authors.lastname: Lichti
authors.forename: Barsam
authors.initials: B
authors.lastname: Mirfattah
authors.forename: Jennifer
authors.initials: J
authors.lastname: Frahm
authors.forename: Carol L
authors.initials: CL
authors.lastname: Nilsson
mesh_terms: D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D030562:Databases, Protein; D054879:Halogenation; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics; D012015:Reference Standards; D057386:Search Engine; D012709:Serum Albumin / Q000737:chemistry / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000592:standards
chemical_list: D010446:Peptide Fragments; D020543:Proteome; D012709:Serum Albumin