Proteomic characterization of carbonylated amino acid sites currently relies on confidently matching tandem mass spectra (MS2) to peptides within a sequence database. Although effective to some degree, reliable proteomic characterization of carbonylated peptides using this approach remains a challenge needing new, complementary solutions. To this end, we developed a method based on partial 18O-labeling of reactive carbonyl modifications, which produces a unique isotope signature in mass spectra of carbonylated peptides and enables their detection without reliance on matching MS2 spectra to a peptide sequence. Key to our method were optimized measures for eliminating trypsin-catalyzed incorporation of 18O at peptide C-termini, and for stabilizing the incorporated 18O within the carbonyl modification to prevent its loss during liquid chromatography separation. Applying our method to a rat skeletal muscle homogenate treated with the carbonyl modification 4-hyroxynonenal (4-HNE), we demonstrated its compatibility with solid-phase hydrazide enrichment of carbonylated peptides from complex mixtures. Additionally, we demonstrated the value of 18O isotope signatures for confirming HNE-modified peptide sequences matched via sequence database searching, and identifying modified peptides missed by MS2 and/or sequence database searching. Combining our 18O-labeling method with a customized automated software script, we systematically evaluated for the first time the efficiency of MS2 and sequence database searching for identifying HNE-modified peptides. We estimated that less than half of the modified peptides selected for MS2 were successfully identified. Collectively, our method and software should provide valuable new tools for investigators studying protein carbonylation via mass spectrometry-based proteomics.
|Original language||English (US)|
|Number of pages||14|
|Journal||Journal of the American Society for Mass Spectrometry|
|State||Published - Jul 2010|
Bibliographical noteFunding Information:
The authors thank the Center for Mass Spectrometry and Proteomics at the University of Minnesota for instrument access and maintenance, in particular Matt Stone for his assistance with operation of the LTQ-Orbitrap. They also thank the Minnesota Supercomputing Institute for maintenance and administration of the SEQUEST cluster and related software. The authors acknowledge funding in part for this work by grant NIA AG017768 (L.V.T.).