UCLA

The advent of top-down protein mass spectrometry (MS), or direct analysis of intact proteins forgoing proteolysis, has transformed the field of protein mass spectrometry, ushering in a new era of protein identification and characterization together with a new set of challenges. The analysis of intact proteins and their direct fragmentation in tandem (MS/MS) mode helps overcome the “inference” problem associated with peptide-based bottom-up proteomics; that is, correctly assigning given peptide fragments and their modifications to the intact protein from which they originated. Despite its many advantages, however, the top-down approach requires extensive sample fractionation and suffers from low sensitivity but much progress has been made. From recently-developed cross-linked polyacrylamide gels, from which intact proteins can be more easily recovered, to the discovery of reagents that enhance protein charging in electrospray ionization (ESI), there have been considerable gains in detection and sensitivity, offering the potential for a more complete and accurate characterization of a “proteoform”: the full complement of the combinatorial possibilities that could arise from a given gene product.

Top-down MS also includes the study of proteins in their native or native-like states. This is especially important in characterizing disease-related proteins, particularly in the context of protein aggregation. Native MS, using electron-capture dissociation (ECD) and ion mobility spectrometry (IMS), enables the study of protein-inhibitor complexes in the gas phase, offering structural insight into stoichiometry, site of inhibitor binding and mechanism of inhibition. In addition, intact analysis and electron-based fragmentation enable the detection of thermally-labile post-translational modifications like phosphorylation, known to play key regulatory roles in shifting proteins towards cytotoxic states. Top-down method developments in protein recovery, separation and supercharging have led to improvements in detection and sensitivity, while top-down MS applications to structural characterization of disease-related proteins have shed more light on the mechanisms of cytotoxic aggregation, offering greater promise of therapeutic development.