Generating Sample-Specific Databases for Mass Spectrometry-Based Proteomic Analysis by Using RNA Sequencing

Mass spectrometry-based methods allow for the direct, comprehensive analysis of expressed proteins and their quantification among different conditions. However, in general identification of proteins by assigning experimental mass spectra to peptide sequences of proteins relies on matching mass spectra to theoretical spectra derived from genomic databases of organisms. This conventional approach limits the applicability of proteomic methodologies to species for which a genome reference sequence is available. Recently, RNA-sequencing (RNA-Seq) became a valuable tool to overcome this limitation by de novo construction of databases for organisms for which no DNA sequence is available, or by refining existing genomic databases with transcriptomic data. Here we present a generic pipeline to make use of transcriptomic data for proteomics experiments. We show in particular how to efficiently fuel proteomic analysis workflows with sample-specific RNA-sequencing databases. This approach is useful for the proteomic analysis of so far unsequenced organisms, complex microbial metatranscriptomes/metaproteomes (for example in the human body), and for refining current proteomics data analysis that solely relies on the genomic sequence and predicted gene expression but not on validated gene products. Finally, the approach used in the here presented protocol can help to improve the data quality of conventional proteomics experiments that can be influenced by genetic variation or splicing events.