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Identification of proteoforms, the different forms of a protein, is important to understand biological processes. A proteoform family is the set of different proteoforms from the same gene. We previously developed the software program Proteoform Suite, which constructs proteoform families and identifies proteoforms by intact-mass analysis. Here, we have applied this approach to top-down proteomic data acquired at the National High Magnetic Field Laboratory 21 tesla Fourier transform ion cyclotron resonance mass spectrometer (data available on the MassIVE platform with identifier MSV000085978). We explored the ability to construct proteoform families and identify proteoforms from the high mass accuracy data that this instrument provides for a complex cell lysate sample from the MCF-7 human breast cancer cell line. There were 2830 observed experimental proteforms, of which 932 were identified, 44 were ambiguous, and 1854 were unidentified. Of the 932 unique identified proteoforms, 766 were identified by top-down MS2 analysis at 1% false discovery rate (FDR) using TDPortal, and 166 were additional intact-mass identifications (∼4.7% calculated global FDR) made using Proteoform Suite. We recently published a proteoform level schema to represent ambiguity in proteoform identifications. We implemented this proteoform level classification in Proteoform Suite for intact-mass identifications, which enables users to determine the ambiguity levels and sources of ambiguity for each intact-mass proteoform identification.

Prefractionation of complex mixtures of proteins derived from biological samples is indispensable for proteome analysis via top-down mass spectrometry (MS). Polyacrylamide gel electrophoresis (PAGE), which enables high-resolution protein separation based on molecular size, is a widely used technique in biochemical experiments and has the potential to be useful in sample fractionation for top-down MS analysis. However, the lack of a means to efficiently recover the separated proteins in-gel has always been a barrier to its use in sample prefractionation. In this study, we present a novel experimental workflow, called Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS ("PEPPI-MS"), which allows top-down MS of PAGE-separated proteins. The optimization of Coomassie brilliant blue staining followed by the passive extraction step in the PEPPI-MS workflow enabled the efficient recovery of proteins, separated on commercial precast gels, from a wide range of molecular weight regions in under 10 min. Two-dimensional separation combining offline PEPPI-MS with online reversed-phase liquid chromatographic separation resulted in identification of over 1000 proteoforms recovered from the target region of the gel (≤50 kDa). Given the widespread availability and relatively low cost of traditional sodium dodecyl sulfate (SDS)-PAGE equipment, the PEPPI-MS workflow will be a powerful prefractionation strategy for top-down proteomics.