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 E. coli ribosome
as a model system of limited complexity. We used deep top-down analysis
to construct an E. coli ribosomal Proteoform
Atlas containing 2099 proteoforms from 52 of the 54 proteins that
make up the E. coli ribosome. We show
that using the Atlas enables confident MS1-only identifications of E. coli ribosomal proteoforms from E. coli 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.