Unipept next: expanding the scope beyond tryptic peptides for advanced microbiome analysis

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 immune responses. These peptides are not restricted to tryptic forms and exhibit a diverse range of lengths and cleavage patterns.

The current limitation of Unipept to match only tryptic peptides constrains its utility in the use-cases described above. 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 protein samples. We validated the performance and correctness of Unipept Next using six datasets from the SIHUMI study, demonstrating its massively enhanced performance and versatility (see Figure 1).