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Targeted 18O-labeling for improved proteomic analysis of carbonylated peptides by mass spectrometry.

ABSTRACT: Proteomic characterization of carbonylated amino acid sites currently relies on confidently matching tandem mass spectra (MS(2)) 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 (18)O-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 MS(2) spectra to a peptide sequence. Key to our method were optimized measures for eliminating trypsin-catalyzed incorporation of (18)O at peptide C-termini, and for stabilizing the incorporated (18)O 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 (18)O isotope signatures for confirming HNE-modified peptide sequences matched via sequence database searching, and identifying modified peptides missed by MS(2) and/or sequence database searching. Combining our (18)O-labeling method with a customized automated software script, we systematically evaluated for the first time the efficiency of MS(2) and sequence database searching for identifying HNE-modified peptides. We estimated that less than half of the modified peptides selected for MS(2) were successfully identified. Collectively, our method and software should provide valuable new tools for investigators studying protein carbonylation via mass spectrometry-based proteomics.

SUBMITTER: Roe MR

PROVIDER: S-EPMC4100935 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Targeted 18O-labeling for improved proteomic analysis of carbonylated peptides by mass spectrometry.

Roe Mikel R MR McGowan Thomas F TF Thompson LaDora V LV Griffin Timothy J TJ

Journal of the American Society for Mass Spectrometry 20100329 7

Proteomic characterization of carbonylated amino acid sites currently relies on confidently matching tandem mass spectra (MS(2)) 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 (18)O-labeling of reactive carbonyl modifications, which produces a unique isotope signature in mass spec ...[more]

PMID: 20434358

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Project description:BackgroundQuantitative measurements of specific protein phosphorylation sites, as presented here, can be used to investigate signal transduction pathways, which is an important aspect of cell dynamics. The presented method quantitatively compares peptide abundances from experiments using 18O/16O labeling starting from elaborated MS spectra. It was originally developed to study signaling cascades activated by amyloid-beta treatment of neurons used as a cellular model system with relevance to Alzheimer's disease, but is generally applicable.ResultsThe presented method assesses, in complete cell lysates, the degree of phosphorylation of specific peptide residues from MS spectra using 18O/16O labeling. The abundance of each observed phospho-peptide from two cell states was estimated from three overlapping isotope contours. The influence of peptide-specific labeling efficiency was removed by performing a label swapped experiment and assuming that the labeling efficiency was unchanged upon label swapping. Different degrees of phosphorylation were reported using the fold change measure which was extended with a confidence interval found to reflect the quality of the underlying spectra. Furthermore a new way of method assessment using simulated data is presented. Using simulated data generated in a manner mimicking real data it was possible to show the method's robustness both with increasing noise levels and with decreasing labeling efficiency.ConclusionThe fold change error assessable on simulated data was on average 0.16 (median 0.10) with an error-to-signal ratio and labeling efficiency distributions similar to the ones found in the experimentally observed spectra. Applied to experimentally observed spectra a very good match was found to the model (<10% error for 85% of spectra) with a high degree of robustness, as assessed by data removal. This new method can thus be used for quantitative signal cascade analysis of total cell extracts in a high throughput mode.

Dataset Information

Targeted 18O-labeling for improved proteomic analysis of carbonylated peptides by mass spectrometry.

Publications

Targeted 18O-labeling for improved proteomic analysis of carbonylated peptides by mass spectrometry.

Similar Datasets

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