Utilizing RNA-Seq data to improve proteomics

Shotgun proteomics utilizes a database search strategy to compare detected mass spectra to a library of theoretical spectra derived from reference genome information. As such, the robustness of proteomics results is contingent upon the completeness and accuracy of the gene annotation in the reference genome. For animal models of disease where genomic annotation is incomplete, such as non-human primates, proteogenomic methods can improve the detection of proteins by incorporating transcriptional data from RNA-Seq to improve proteomics search databases used for peptide spectral matching. Customized search databases derived from RNA-Seq data are capable of identifying unannotated genetic and splice variants while simultaneously reducing the number of comparisons to only those transcripts actively expressed in the tissue.

Texas Biomedical Research Institute scientists collected RNA-Seq and proteomic data from 10 vervet monkey liver samples and used the RNA-Seq data to curate sample-specific search databases which were analyzed in the program Morpheus. They compared these results against those from a search database generated from the reference vervet genome. A total of 284 previously unannotated splice junctions were predicted by the RNA-Seq data, 92 of which were confirmed by peptide spectral matches. More than half (53/92) of these unannotated splice variants had orthologs in other non-human primates, suggesting that failure to match these peptides in the reference analyses likely arose from incomplete gene model information. The sample-specific databases also identified 101 unique peptides containing single amino acid substitutions which were missed by the reference database. Because the sample-specific searches were restricted to actively expressed transcripts, the search databases were smaller, more computationally efficient, and identified more peptides at the empirically derived 1 % false discovery rate.

Descriptive statistics for the RNA-Seq and mass spectrometry analyses utilizing the Vervet reference search database (REFdb, 19,255 gene entries) and the sample-specific databases (SSdb)

Proteogenomic approaches are ideally suited to facilitate the discovery and annotation of proteins in less widely studies animal models such as non-human primates. The researchers expect that these approaches will help to improve existing genome annotations of non-human primate species such as vervet.