The analysis of post translational modifications (PTM’s) on proteins and peptides is a highly sought after technique by biologists trying to dissect signal transduction pathways or identify modifications that could be a biomarker for disease. In most cases PTM analysis by conventional proteomics techniques such as LCMS with CID is challenging. Many modifications such as phosphorylation, sulphation, glycation, O-glycosylation are labile in tandem mass spectrometry leading to ambiguity in site assignment from MSMS spectra using search engines. Electron Capture Dissociation and Electron Transfer Dissociation do preserve these labile modifications, but traditionally these techniques are slow with long reaction times, meaning that only a few precursors are fragmented per second in LCMS experiments.
Electron Activated Dissociation (EAD) on the ZenoTOF 7600 system is a fast electron based collision cell that is tuneable from ECD like energies all the way up to high energy electron reactions (EIEIO) similar to the energies used in electron impact ionisation. For peptide PTM’s ECD like conditions are used on the ZenoTOF 7600 with reaction times of only 10ms, giving the capability of performing EAD in LCMS runs at >30Hz. This acquisition speed is on a par with CID and totally compatible with the analysis of complex mixtures with short gradients.
Data analysis of these EAD spectra requires a search engine that utilises the c and z ions found in the EAD MSMS spectra. Mascot, from MatrixScience, works perfectly with these spectra and has been successfully used in phosphoproteomic studies, glycation, glycosylation and other labile PTM’s. Examples will be shown and the clean MSMS spectra acquired using EAD on the ZenoTOF 7600 system allows PTM site assignment with very high confidence.