Proteomic mapping of 4-hydroxynonenal protein modification sites by solid-phase hydrazide chemistry and mass spectrometry

Mikel R. Roe, Hongwei Xie, Sricharan Bandhakavi, Timothy J. Griffin

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)3747-3756
Number of pages10
JournalAnalytical chemistry
Volume79
Issue number10
DOIs
StatePublished - May 15 2007

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Mass spectrometry
Amino Acids
Yeast
Proteins
Ions
Basic Amino Acids
Liquid chromatography
Proteome
Complex Mixtures
Cell culture
Histidine
Aldehydes
Peptides
Proteomics
4-hydroxy-2-nonenal

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Proteomic mapping of 4-hydroxynonenal protein modification sites by solid-phase hydrazide chemistry and mass spectrometry. / Roe, Mikel R.; Xie, Hongwei; Bandhakavi, Sricharan; Griffin, Timothy J.

In: Analytical chemistry, Vol. 79, No. 10, 15.05.2007, p. 3747-3756.

Research output: Contribution to journalArticle

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