A modified database search strategy leads to improved identification of in vitro brominated peptides spiked into a complex proteomic sample
(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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- author
- Liu, Huiling ; Lichti, Cheryl F ; Mirfattah, Barsam ; Frahm, Jennifer and Nilsson, Carol L LU
- publishing date
- 2013-09-06
- type
- 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 (ACS)
- external identifiers
-
- pmid:23898862
- 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
- 2024-06-23 17:16:11
@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}},
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}},
language = {{eng}},
month = {{09}},
number = {{9}},
pages = {{54--4248}},
publisher = {{The American Chemical Society (ACS)}},
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}},
doi = {{10.1021/pr400472c}},
volume = {{12}},
year = {{2013}},
}