Expanding Proteoform Identifications in Top-Down Proteomic Analyses by Constructing Proteoform Families

In top-down proteomics,
intact proteins are analyzed by tandem
mass spectrometry and proteoforms, which are defined forms of a protein
with specific sequences of amino acids and localized post-translational
modifications, are identified using precursor mass and fragmentation
data. Many proteoforms that are detected in the precursor scan (MS1)
are not selected for fragmentation by the instrument and therefore
remain unidentified in typical top-down proteomic workflows. Our laboratory
has developed the open source software program Proteoform Suite to
analyze MS1-only intact proteoform data. Here, we have adapted it
to provide identifications of proteoform masses in precursor MS1 spectra
of top-down data, supplementing the top-down identifications obtained
using the MS2 fragmentation data. Proteoform Suite performs mass calibration
using high-scoring top-down identifications and identifies additional
proteoforms using calibrated, accurate intact masses. Proteoform families,
the set of proteoforms from a given gene, are constructed and visualized
from proteoforms identified by both top-down and intact-mass analyses.
Using this strategy, we constructed proteoform families and identified
1861 proteoforms in yeast lysate, yielding an approximately 40% increase
over the original 1291 proteoform identifications observed using traditional
top-down analysis alone.