Full Peptide Sequence Coverage and Unambiguous Phosphorylation Site Localization by EThcD

[Orbitrap_SciLib]

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 [1] 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.[2] 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.[3] Data was analyzed using the SEQUEST algorithm embedded in Proteome Discoverer (Thermo Fisher Scientific, Bremen). Results: • EThcD generates dual ion series and provides for data-rich MS/MS spectra • Average peptide sequence coverage in EThcD is about 95% • EThcD generates extensive sequence information that facilitates sensitive phosphosite localization


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