Journal
JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY
Volume 27, Issue 8, Pages 1322-1327Publisher
SPRINGER
DOI: 10.1007/s13361-016-1403-3
Keywords
Peptide modification; Hydroxyl radical protein footprinting; Fast photochemical oxidation of proteins; Size exclusion chromatography; Electron transfer dissociation
Funding
- National Institute of General Medical Sciences [R01 GM096049, P41 GM103390]
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One difficult problem in the analysis of peptide modifications is quantifying isomeric modifications that differ by the position of the amino acid modified. HPLC separation using C18 reverse phase chromatography coupled with electron transfer dissociation (ETD) in tandem mass spectrometry has recently been shown to be able to relatively quantify how much of a given modification occurs at each amino acid position for isomeric mixtures; however, the resolution of reverse phase chromatography greatly complicates quantification of isomeric modifications by ETD because of the chromatographic separation of peptides with identical modifications at different sequence positions. Using peptide oxidation as a model system, we investigated the use of size exclusion chromatography coupled with ETD fragmentation to separate peptide sequences. This approach allows for the benefits of chromatographic separation of peptide sequences while ensuring co-elution of modification isomers for accurate relative quantification of modifications using standard data-dependent acquisitions. Using this method, the relative amount of modification at each amino acid can be accurately measured from single ETD MS/MS spectra in a standard data-dependent acquisition experiment.
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