Unipept (https://doi.org/10.1021/acs.jproteome.8b00716), a pioneering tool in metaproteomics, has significantly advanced the analysis of complex ecosystems by facilitating both taxonomic and functional insights from environmental samples. Initially, 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 metaproteomics, samples are typically prepared using trypsin which cleaves proteins at specific cleavage sites (https://doi.org/10.1038/s41467-021-27542-8). Occasionally, trypsin skips a cleavage site, resulting in fragments composed of multiple tryptic peptides, referred to as peptides with missed cleavages. Semi-tryptic peptides, produced when one terminus is generated by trypsin cleavage and the other is not, were previously undetectable by Unipept. Accurately identifying semi-tryptic peptides is crucial for understanding the complex proteolytic activities of the gut microbiome (https://doi.org/10.1186/s40168-020-00967-x).

Immunopeptidomics (https://doi.org/10.1016/j.smim.2023.101727), an upcoming research discipline, focuses on characterizing HLA-peptides presented by MHC molecules, which are vital to study our immune responses. In the context of immunopeptidomics, the peptides presented by MHC molecules are typically between 8 and 25 amino acids long (depending on the class of the MHC molecule) and are derived from both self and non-self (e.g. viral or bacterial) proteins. By extracting the peptides presented by MHC molecules and identifying the organism they originate from, researchers can gain a deep understanding of how the immune system recognizes and responds to pathogens, tumors and other abnormal cells, and eventually help to identify peptide biomarkers for diseases.

The past limitation of Unipept to match only tryptic peptides constrained its utility in characterizing HLA-peptides, studied by immunopeptidomics researchers. Over the past year, we have integrated a range of sophisticated index structures from the field of metagenomics, such as Enhanced Suffix Arrays (ESA), FM-indices, and the R-index, to enhance Unipept's capability in identifying substrings in large text bodies efficiently.

Unipept Next is the next-generation of Unipept, capable of analyzing peptides regardless of how their originating proteins were cleaved. Utilizing an Enhanced Suffix Array, Unipept Next can identify the proteins from which a peptide originates in milliseconds, enabling rapid taxonomic and functional analysis of peptide samples.