Oktoberfest: Open-source spectral library generation and rescoring pipeline based on Prosit.

Machine learning (ML) and deep learning (DL) models for peptide property prediction such as Prosit have enabled the creation of high quality in silico reference libraries. These libraries are used in various applications, ranging from data-independent acquisition (DIA) data analysis to data-driven rescoring of search engine results. Here, we present Oktoberfest, an open source Python package of our spectral library generation and rescoring pipeline originally only available online via ProteomicsDB. Oktoberfest is largely search engine agnostic and provides access to online peptide property predictions, promoting the adoption of state-of-the-art ML/DL models in proteomics analysis pipelines. We demonstrate its ability to reproduce and even improve our results from previously published rescoring analyses on two distinct use cases. Oktoberfest is freely available on GitHub (https://github.com/wilhelm-lab/oktoberfest) and can easily be installed locally through the cross-platform PyPI Python package.

Predicting fragment intensities and retention time of iTRAQ- and TMTPro-labeled peptides with Prosit-TMT.

Isobaric labeling increases the throughput of proteomics by enabling the parallel identification and quantification of peptides and proteins. Over the past decades, a variety of isobaric tags have been developed allowing the multiplexed analysis of up to 18 samples. However, experiments utilizing such tags often exhibit reduced identification rates and thus show decreased analytical depth. Re-scoring has been shown to rescue otherwise missed identifications but was not yet systematically applied on isobarically labeled data. Because iTRAQ 4/8-plex and the recently released TMTpro 16/18-plex share similar characteristics with TMT 6/10/11-plex, we hypothesized that Prosit-TMT, trained exclusively on 6/10/11-plex labeled peptides, may be applicable to these isobaric labeling strategies as well. To investigate this, we re-analyzed nine publicly available datasets covering iTRAQ and TMTpro labeling for samples with human and mouse origin. We highlight that Prosit-TMT shows remarkably good performance when comparing experimentally acquired and predicted fragmentation spectra (R of 0.84 - 0.9) and retention times (ΔRT95% of 3% - 10% gradient time) of peptides. Furthermore, re-scoring substantially increases the number of confidently identified spectra, peptides, and proteins.