Intact Mass Proteomics\nUsing a Proteoform Atlas

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