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A modified database search strategy leads to improved identification of in vitro brominated peptides spiked into a complex proteomic sample

Liu, Huiling; Lichti, Cheryl F; Mirfattah, Barsam; Frahm, Jennifer and Nilsson, Carol L LU (2013) In Journal of Proteome Research 12(9). p.54-4248
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... (More)

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.

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Contribution to journal
publication status
published
keywords
Amino Acid Sequence, Cell Line, Tumor, Databases, Protein, Halogenation, Humans, Molecular Sequence Data, Peptide Fragments, Peptide Mapping, Protein Processing, Post-Translational, Proteome, Proteomics, Reference Standards, Search Engine, Serum Albumin, Tandem Mass Spectrometry, Journal Article, Research Support, Non-U.S. Gov't
in
Journal of Proteome Research
volume
12
issue
9
pages
7 pages
publisher
The American Chemical Society
external identifiers
  • scopus:84883813458
ISSN
1535-3893
DOI
10.1021/pr400472c
language
English
LU publication?
no
id
0a2007bd-5a18-40df-afee-4cca4e6d101f
date added to LUP
2017-05-16 10:27:24
date last changed
2019-02-20 10:36:00
@article{0a2007bd-5a18-40df-afee-4cca4e6d101f,
  abstract     = {<p>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.</p>},
  author       = {Liu, Huiling and Lichti, Cheryl F and Mirfattah, Barsam and Frahm, Jennifer and Nilsson, Carol L},
  issn         = {1535-3893},
  keyword      = {Amino Acid Sequence,Cell Line, Tumor,Databases, Protein,Halogenation,Humans,Molecular Sequence Data,Peptide Fragments,Peptide Mapping,Protein Processing, Post-Translational,Proteome,Proteomics,Reference Standards,Search Engine,Serum Albumin,Tandem Mass Spectrometry,Journal Article,Research Support, Non-U.S. Gov't},
  language     = {eng},
  month        = {09},
  number       = {9},
  pages        = {54--4248},
  publisher    = {The American Chemical Society},
  series       = {Journal of Proteome Research},
  title        = {A modified database search strategy leads to improved identification of in vitro brominated peptides spiked into a complex proteomic sample},
  url          = {http://dx.doi.org/10.1021/pr400472c},
  volume       = {12},
  year         = {2013},
}