Deep profiling and custom databases improve detection of proteoforms generated by alternative splicing

Alternative pre-mRNA splicing has long been proposed to greatly contribute to proteome complexity. However, the extent to which mature mRNA isoforms are successfully translated into protein remains controversial. Here, we used high-throughput RNA sequencing and mass spectrometry (MS)-based proteomics to better evaluate the translation of alternatively spliced mRNAs. To increase proteome coverage and improve protein quantitation, we optimized cell fractionation and sample processing steps at both the protein and peptide level. Furthermore, we generated a custom spectral library trained on analysis of RNA-Seq data with MAJIQ, an algorithm optimized to detect differential and unannotated junction usage for a given splice site. Using this custom library to match against tandem mass spectra acquired by data independent acquisition (DIA), we improved identification of splicing-derived proteoforms by ~30% as compared to use of the SwissProt database alone. Moreover, our increased depth and detection of proteins allowed us to track changes in the transcriptome and proteome induced by T cell stimulation, as well as fluctuations in protein sub-cellular localization. In sum, our data here confirms that use of generic databases in proteomic studies under-estimates the number of spliced mRNA isoforms that are translated into protein and provides a workflow that improves isoform detection in large-scale proteomic experiments.