Activated-Ion Electron Transfer Dissociation Improves the Ability of Electron Transfer Dissociation to Identify Peptides in a Complex Mixture

Using a modified electron transfer dissociation (EID)-enabled quadrupole linear ion trap (QLT) mass spectrometer, we demonstrate the utility of IR activation concomitant with ETD ion-ion reactions (activated-ion ETD, AI-ETD). Analyzing 12 strong cation exchanged (SCX) fractions of a LysC digest of human cell protein extract using LID, collision-activated dissociation (CAD), and AI-ETD, we find that AI-ETD generates 13 405 peptide spectral matches (PSMs) at a 1% false-discovery rate (1% FDR), surpassing both ETD (7 968) and CAD (10 904). We also analyze 12 SCX fractions of a tryptic digest of human cell protein extract and find that E) produces 6 234 PSMs, AI-ETD 9 130 PSMs, and CAD 15 209 PSMs. Compared to ELD with supplemental collisional activation (ETcaD), AI-ETD generates similar to 80% more PSMs for the whole cell lysate digested with trypsin and similar to 50% more PSMs for the whole cell lysate digested with LysC.