Proteoform-predictor: Increasing the Phylogenetic Reach of Top-Down Proteomics

Proteoforms are distinct molecular forms of proteins
that act as
building blocks of organisms, with post-translational modifications
(PTMs) being one of the key changes that generate these variations.
Mass spectrometry (MS)-based top-down proteomics (TDP) is the leading
technology for proteoform identification due to its preservation of
intact proteoforms for analysis, making it well-suited for comprehensive
PTM characterization. A crucial step in TDP is searching MS data against
a database of candidate proteoforms. To extend the reach of TDP to
organisms with limited PTM annotations, we developed Proteoform-predictor, an open-source tool that integrates homology-based PTM site prediction
into proteoform database creation. The new tool creates databases
of proteoform candidates after registration of homologous sequences,
transferring PTM sites from well-characterized species to those with
less comprehensive proteomic data. Our tool features a user-friendly
interface and intuitive workflow, making it accessible to a wide range
of researchers. We demonstrate that Proteoform-predictor expands
proteoform databases with tens of thousands of proteoforms for three
bacterial strains by comparing them to the reference proteome of Escherichia coli (E. coli) K12. Subsequent TDP analysis for Serratia marcescens (S. marcescens) and Salmonella typhimurium (S. typhimurium) demonstrated significant improvement in protein and proteoform
identification, even for proteins with variant sequences. As TDP technology
advances, Proteoform-predictor will become an important tool
for expanding the applicability of proteoform identification and PTM
biology to more diverse species across the phylogenetic tree of life.