Abstract

Proteins of unknown function represent a significant gap in our understanding of biological processes, encompassing large portions of the proteomes of many organisms, especially prokaryotes. Addressing this gap is critical to understanding the biology and pathogenicity of such organisms. We introduce ProtPen, an open-source pipeline that facilitates protein function prediction by combining eggNOG-mapper for sequence-based annotation with Foldseek for rapid structural similarity searches using AlphaFold-predicted protein structures. Annotation results from both tools are merged and enriched with UniProt metadata to produce a comprehensive output suitable for downstream analysis. The pipeline requires only a FASTA input file with UniProt identifiers, and is designed to analyze datasets on the scale of whole proteomes. Benchmarking on a curated dataset of well-characterized Pseudomonas aeruginosa proteins demonstrated an annotation accuracy of >90%, and highlighted the complementarity of sequence- and structure-based methods. Further evaluation of ProtPen included its application to biologically relevant datasets, comprising proteins of unknown function that exhibited significant differential abundances in a proteomics dataset of P. aeruginosa, and uncharacterized glycoproteins from Haloferax volcanii. ProtPen is readily extensible to incorporate additional protein function prediction tools. In summary, this pipeline facilitates the systemwide annotation of proteins of unknown function from proteomic datasets and whole proteomes.