PROTEOFORMER 2.0: Further Developments in the Ribosome Profiling-assisted Proteogenomic Hunt for New Proteoforms*

The PROTEOFORMER pipeline feeds ribosome profiling-driven information into an MS/MS search space. The pipeline has been greatly expanded and updated since its first publication. These novelties are presented and validated with matching MS/MS data, leading to the endorsement of a set of new proteoforms on MS/MS level and to a collection of general considerations for the ribosome profiling-based proteogenomics community. Graphical Abstract Highlights PROTEOFORMER adds ribosome profiling information to MS/MS search spaces. The PROTEOFORMER pipeline is greatly expanded and updated since its first publication. New features are demonstrated with matching ribosome profiling and MS/MS data. Experiments lead to MS/MS-proven proteoforms and general proteogenomic notices. PROTEOFORMER is a pipeline that enables the automated processing of data derived from ribosome profiling (RIBO-seq, i.e. the sequencing of ribosome-protected mRNA fragments). As such, genome-wide ribosome occupancies lead to the delineation of data-specific translation product candidates and these can improve the mass spectrometry-based identification. Since its first publication, different upgrades, new features and extensions have been added to the PROTEOFORMER pipeline. Some of the most important upgrades include P-site offset calculation during mapping, comprehensive data pre-exploration, the introduction of two alternative proteoform calling strategies and extended pipeline output features. These novelties are illustrated by analyzing ribosome profiling data of human HCT116 and Jurkat data. The different proteoform calling strategies are used alongside one another and in the end combined together with reference sequences from UniProt. Matching mass spectrometry data are searched against this extended search space with MaxQuant. Overall, besides annotated proteoforms, this pipeline leads to the identification and validation of different categories of new proteoforms, including translation products of up- and downstream open reading frames, 5′ and 3′ extended and truncated proteoforms, single amino acid variants, splice variants and translation products of so-called noncoding regions. Further, proof-of-concept is reported for the improvement of spectrum matching by including Prosit, a deep neural network strategy that adds extra fragmentation spectrum intensity features to the analysis. In the light of ribosome profiling-driven proteogenomics, it is shown that this allows validating the spectrum matches of newly identified proteoforms with elevated stringency. These updates and novel conclusions provide new insights and lessons for the ribosome profiling-based proteogenomic research field. More practical information on the pipeline, raw code, the user manual (README) and explanations on the different modes of availability can be found at the GitHub repository of PROTEOFORMER: https://github.com/Biobix/proteoformer.

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