Intact Mass Proteomics Using a Proteoform Atlas

Top-down proteomics, the characterization of intact proteoforms by tandem mass spectrometry, is the principal method for proteoform characterization in complex samples. Top-down proteomics relies on precursor isolation and subsequent gas-phase fragmentation to make proteoform identifications. While this strategy can produce highly detailed molecular information, the reliance on time-intensive tandem MS limits the speed with which proteoforms can be identified. We suggest that once proteoforms have been identified by top-down analysis in a system of interest, and archived in a system-specific Proteoform Atlas, subsequent analyses in that system can utilize the Atlas information to enable simpler and faster MS1-only identifications. We explore this idea here, using the <i>E. coli</i> ribosome as a model system of limited complexity. We used deep top-down analysis to construct an <i>E. coli</i> ribosomal Proteoform Atlas containing 2099 proteoforms from 52 of the 54 proteins that make up the <i>E. coli</i> ribosome. We show that using the Atlas enables confident MS1-only identifications of <i>E. coli</i> ribosomal proteoforms from <i>E. coli</i> that were perturbed by exposure to cold. Furthermore, this Atlas strategy identifies proteoforms up to 77% more rapidly compared to top-down identifications that require acquisition of both MS1 and MS2 spectra.