The high-throughput nature of proteomics mass spectrometry is enabled by a productive combination of data acquisition protocols and the computational tools used to interpret the resulting spectra. One of the key components in mainstream protocols is the generation of tandem mass (MS/MS) spectra by peptide fragmentation using collision induced dissociation (CID), the approach currently used in the large majority of proteomics experiments to routinely identify hundreds to thousands of proteins from single mass spectrometry runs. Complementary to these, alternative peptide fragmentation methods such as Electron Capture/Transfer Dissociation (ECD/ETD, respectively) and Higher-energy Collision Dissociation (HCD) have consistently achieved significant improvements in the identification of certain classes of peptides, proteins and post-translational modifications. Recognizing these advantages, mass spectrometry instruments now conveniently support fine-tuned methods that automatically alternate between peptide fragmentation modes for either different types of peptides or for acquisition of multiple MS/MS spectra from each peptide. But while these developments have the potential to substantially improve peptide identification, their routine application requires corresponding adjustments to the software tools and procedures used for automated downstream processing. This review discusses the computational implications of alternative and alternate modes of MS/MS peptide fragmentation and addresses some practical aspects of using such protocols for identification of peptides and post-translational modifications.
The review article is published online in the journal Molecular & Cellular Proteomics and is free to access.