Relative Quantification of Sites of Peptide and Protein Modification using Size Exclusion Chromatography coupled with Electron Transfer Dissociation

摘要

Quantifying peptide modification isomers that differ only by the position of the amino acid modified is a serious challenge in protein analysis. Quantification of these isomers by electron transfer dissociation (ETD) product ion intensities has shown promise. Common practice for proteomics involves HPLC separation prior to ETD fragmentation using C18 reverse phase chromatography; however, the partial chromatographic separation of peptides with identical modifications at different sequence positions greatly complicates quantification of these modification isomers by ETD. Using peptide oxidation as a model system, we investigated the use of size exclusion chromatography coupled with ETD fragmentation to separate peptide sequences while ensuring co-elution of modification isomers for accurate relative quantification of modifications using a standard data-dependent acquisition strategy.