Figure 4.

Comparison of site-determining ions for the sY and pY peptide panel using different fragmentation regimes. Two sample pools were generated containing either the panel of 12 sY- or 12 pY-containing peptides and subjected to LC–MS/MS on the Fusion Lumos Tribrid mass spectrometer (ThermoFisher) using fragmentation conditions as detailed. Ranging NCEs were applied for CID, HCD, ETciD, and EThcD as denoted. The ETD component was charge state-calibrated.⁹⁰ UVPD activation time was either calibrated to molecular weight (%)⁹¹ or manually set (ms). Data were searched with COMET, and the highest scoring PSM was selected for further investigation. The heatmap shows the log₂ ratio of the number of observed MS2 product ions containing the known modification mass shift (Δ80 Da) normalized to the total number of potential PTM-containing product ions (log₂(ion mean + 1). Ions correlating to fragmentation at the same position in the peptide (e.g., a₄/b₄, y₈/y₈²⁺/z₈) were collapsed into a single entry. The modification site is depicted by “[Y]”, and charge states are visualized separately. Green = all potential mass shift containing product ions detected; red = no potential mass shift containing product ions detected; white = no MS/MS spectra were confidently identified.