Proteomic mapping of 4-hydroxynonenal protein modification sites by solid-phase hydrazide chemistry and mass spectrometry.
The modification of proteins by the cytotoxic, reactive aldehyde 4-hydroxynonenal (HNE) is known to alter protein function and impair cellular mechanisms. In order to identify susceptible amino acid sites of HNE modification within complex biological mixtures by microcapillary liquid chromatography and linear ion trap tandem mass spectrometry, we have developed a solid-phase capture and release strategy that utilizes reversible hydrazide chemistry to enrich HNE-modified peptides. To maximize the detection of fragment ions diagnostic of HNE modification, both neutral loss-dependent acquisition of MS/MS/MS spectra and the pulsed Q dissociation operation mode were employed. When the solid-phase hydrazide enrichment strategy was applied to a yeast lysate treated with HNE, 125 distinct amino acid sites of HNE modification were mapped on 67 different proteins. The endogenous susceptibility of many of these proteins to HNE modification was demonstrated by analyzing HNE-treated yeast cell cultures with a complementary biotin hydrazide enrichment strategy. Further analysis revealed that the majority of amino acid sites susceptible to HNE modification were histidine residues, with most of these sites being flanked by basic amino acid residues, and predicted to be solvent exposed. These results demonstrate the effectiveness of this novel strategy as a general platform for proteome-scale identification of amino acid sites susceptible to HNE modification from within complex mixtures.
Roe
Mikel R
MR
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 420 Washington Avenue SE, Minneapolis, Minnesota 55445, USA.
Xie
Hongwei
H
Bandhakavi
Sricharan
S
Griffin
Timothy J
TJ
D000447:Aldehydes / Q000378:metabolism*; D001665:Binding Sites; D005656:Fungal Proteins / Q000032:analysis; D006834:Hydrazines; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D000447:Aldehydes; D005656:Fungal Proteins; D006834:Hydrazines; D011506:Proteins; C027576:4-hydroxy-2-nonenal