Ion Activation Methods for Top‐Down Proteomics

Mass spectrometry (MS) has emerged as a premier method used to characterize the sequences of proteins. Top-down proteomics aims to capture the multiple sources of structural diversity reflected in proteins, such as those that arise from alternative RNA splicing events or the addition of post-translational modifications. Tandem MS (i.e., MS/MS) represents a critical component of a top-down proteomics experiment, as the resulting fragmentation patterns unveil various structural features associated with protein function. This review spotlights recent developments and applications of ion activation methods used to decipher the structural properties of intact proteins, including collisional activation and those based on the use of electrons and photons. The analysis of fragment ions generated by these MS/MS methods are also discussed, along with an outlook on future developments in the field related to instrumentation and burgeoning approaches to top-down proteomics, such as single-cell methods.