Free radicals, electrophiles, and endogenous reactiveintermediates are generated during normal physiologicalprocesses and are capable of modifying DNA, lipids, andproteins. However, elevated levels of oxidative modifications of proteins by reactive species are implicated in theetiology and pathology of oxidative stress-mediated diseases, neurodegeneration, and aging. A mass spectrometry-based approach is reported that aids to the identificationand characterization of carbonyl-modified proteins. Themethod uses
N'-aminooxymethylcarbonylhydrazino
D-biotin, a biotinylated hydroxylamine derivative that formsan oxime derivative with the aldehyde/keto group foundin oxidatively modified proteins. In this paper, the methodis demonstrated for one class of carbonyl-modified proteins, namely, oxylipid peptide and protein conjugatesformed by
Michael addition-type conjugation reactions of
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,
![](/images/gifchars/beta2.gif)
-unsaturated aldehydic lipid peroxidation productswith nucleophilic peptide side chains. This new application of an "old" probe, which has been used for the detection of abasic sites in DNA strands, introduces a biotinmoiety into the oxylipid peptide conjugate. The biotin-modified oxylipid peptide conjugate is then amenable toenrichment using avidin affinity capture. The describedmethod represents an attractive alternative to hydrazine-based derivatization methods for oxidized peptides andproteins because the reduction step necessary for thetransformation of the hydrazone bond to the chemicallymore stable hydrazine bond can be omitted. Tandem massspectrometry of the labeled oxylipid peptide conjugatesindicates that the biotin moiety is at least partially retainedon the fragment ion during the collisionally induceddissociation experiments, a prerequisite for the use ofautomated database searching of uninterpreted tandemmass spectra. The reported approach is outlined for thedetection, identification, and characterization of oxylipidpeptide conjugates, but the labeling chemistry may alsobe applicable to other carbonyl-modified proteins.