PROTEOFORMER

PROTEOFORMER enhances mass spectrometry (MS) --based proteomics studies by integrating mRNA sequencing data to expand the translation product search space. Recognizing the challenges associated with the direct use of mRNA-seq data for constructing protein sequence search databases—such as the complex regulation of mRNA translation and the necessity for three—or six-frame translation—PROTEOFORMER leverages ribosome profiling. This method involves sequencing ribosome-protected mRNA fragments, offering a genome-wide visualization of ribosome occupancy across the transcriptome.

The translation initiation site calling algorithm facilitates the precise delineation of open reading frames (ORFs) for all translation products, allowing for compiling a comprehensive protein synthesis-based sequence database. This database is tailored for MS-based identification, significantly enhancing protein identification rates. Specifically, using PROTEOFORMER has increased overall protein identification rates by 3% and 11% for human and mouse samples, respectively.

PROTEOFORMER enables the proteome-wide detection of various proteoforms, including 5'-extended proteoforms, upstream ORF translation, and near-cognate translation start sites. These capabilities highlight the tool's potential in uncovering the proteome's complexity beyond what is traditionally detectable, offering insights into protein diversity and the regulatory mechanisms of translation.