Unipept 6.0: Expanding metaproteomics analysis with support for missed cleavages, semi-tryptic and non-tryptic peptides

Unipept, a pioneering software tool in metaproteomics, has significantly advanced the analysis of complex ecosystems by facilitating both taxonomic and functional insights from environmental samples. From the onset, Unipept’s capabilities focused on tryptic peptides, utilizing the predictability and consistency of trypsin digestion to efficiently construct a protein reference database. However, the evolving landscape of proteomics and emerging fields like immunopeptidomics necessitate a more versatile approach that extends beyond the analysis of tryptic peptides. In this article, we present Unipept 6.0, featuring a novel protein reference database, powered by a Sparse Suffix Array index. This advancement enables the analysis of semi-tryptic peptides, peptides with missed cleavages, and non-tryptic peptides such as those from immunopeptidomics (e.g. MHC- and HLA-peptides). A benchmark study demonstrates that this version of Unipept’s performance matches or exceeds that of previous versions, while significantly expanding its application range.