A Modified Database Search Strategy Leads to Improved
Identification of in Vitro Brominated Peptides Spiked into a Complex
Proteomic Sample
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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 <sup>81</sup>Br rather than <sup>79</sup>Br for dibromotyrosine (<sup>79</sup>Br<sup>81</sup>BrY), 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