The field of proteomics almost uniformly relies on peptide cation analysis, leading to an underrepresentation of acidic portions of proteomes, including relevant acidic post-translational modifications. Despite the many benefits negative mode proteomics can offer, peptide anion analysis remains in its infancy, due mainly to challenges with high pH reversed phase separations and a lack of robust fragmentation methods suitable for peptide anion characterization.
Electron transfer dissociation (ETD) has been broadly adopted and is now available on a variety of commercial mass spectrometers. Unlike collisional activation techniques, optimal performance of ETD requires considerable user knowledge and input. ETD reaction duration is one key parameter that can greatly influence spectral quality and overall experiment outcome. We describe a calibration routine that determines the correct number of reagent anions necessary to reach a defined ETD reaction rate.