on 07-23-201302:39 AM - edited on 10-15-202111:38 AM by Closed Account
Frese CK, Nolting D, van den Toorn H, Taus T, Zhou H, Altelaar AFM, Griep-Raming J, Mechtler K, Heck AJR, Mohammed S ASMS 2013 Poster Note Introduction Electron Transfer Dissociation (ETD) efficiency is low for doubly and triply charged peptide precursors. Supplemental activation of the charge-reduced precursor by resonant-excitation CID  increases the yield of c/z- ions. However, the unreacted precursor ions typically remain the most abundant species after ETD. We reasoned that beam-type collision induced dissociation can be utilized to fragment the unreacted and the charge-reduced precursor simultaneously in order to generate extensive peptide backbone fragmentation.
Methods: Control software of an Orbitrap Velos (Thermo Fisher Scientific, Bremen) was modified to allow HCD all-ion fragmentation following an initial ETD reaction in the LTQ. Human cell lysate was digested with trypsin and Lys-N, respectively, and fractionated by low-pH strong cation exchange (SCX). Phosphopeptide enrichment was performed using Ti4+-IMAC. Data was analyzed using the SEQUEST algorithm embedded in Proteome Discoverer (Thermo Fisher Scientific, Bremen).
• EThcD generates dual ion series and provides for
data-rich MS/MS spectra
• Average peptide sequence coverage in EThcD is
• EThcD generates extensive sequence information
that facilitates sensitive phosphosite localization