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

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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; Nilsson, Carol L.

In: Journal of Proteome Research, Vol. 12, No. 9, 06.09.2013, p. 4248-54.

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T1 - A modified database search strategy leads to improved identification of in vitro brominated peptides spiked into a complex proteomic sample

AU - Liu, Huiling

AU - Lichti, Cheryl F

AU - Mirfattah, Barsam

AU - Frahm, Jennifer

AU - Nilsson, Carol L

PY - 2013/9/6

Y1 - 2013/9/6

N2 - 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.

AB - 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.

KW - Amino Acid Sequence

KW - Cell Line, Tumor

KW - Databases, Protein

KW - Halogenation

KW - Humans

KW - Molecular Sequence Data

KW - Peptide Fragments

KW - Peptide Mapping

KW - Protein Processing, Post-Translational

KW - Proteome

KW - Proteomics

KW - Reference Standards

KW - Search Engine

KW - Serum Albumin

KW - Tandem Mass Spectrometry

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1021/pr400472c

DO - 10.1021/pr400472c

M3 - Article

VL - 12

SP - 4248

EP - 4254

JO - Journal of Proteome Research

T2 - Journal of Proteome Research

JF - Journal of Proteome Research

SN - 1535-3893

IS - 9

ER -