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Full Peptide Sequence Coverage and Unambiguous Phosphorylation Site Localization by EThcD

Reputable Mentor II
Reputable Mentor II
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

Thermo Fisher Scientific
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Last update:
‎10-15-2021 11:38 AM
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