Integrated proteogenomics database

When using the iPtgxDB web service, please cite

U. Omasits, A. R. Varadarajan, M. Schmid, S. Goetze, D. Melidis, M. Bourqui, O. Nikolayeva, M. Quebatte, A. Patrignani, C. Dehio, J. E. Frey, M. D. Robinson, B. Wollscheid, and C. H. Ahrens

An integrative strategy to identify the entire protein coding potential of prokaryotic genomes by proteogenomics

Genome Research, 27 , 2083-2095, 2017

Accurate annotation of all protein-coding sequences (CDSs) is an essential prerequisite to fully exploit the rapidly growing repertoire of completely sequenced prokaryotic genomes. However, large discrepancies among the number of CDSs annotated by different resources, missed functional short open reading frames (sORFs), and overprediction of spurious ORFs represent serious limitations. Our strategy towards accurate and complete genome annotation consolidates CDSs from multiple reference annotation resources, *ab initio* gene prediction algorithms and *in silico* ORFs (a modified six-frame translation considering alternative start codons) in an integrated proteogenomics database (iPtgxDB) that covers the entire protein-coding potential of a prokaryotic genome. By extending the PeptideClassifier concept of unambiguous peptides for prokaryotes, close to 95% of the identifiable peptides imply one distinct protein, largely simplifying downstream analysis. Searching a comprehensive Bartonella henselae proteomics dataset against such an iPtgxDB allowed us to unambiguously identify novel ORFs uniquely predicted by each resource, including lipoproteins, differentially expressed and membrane-localized proteins, novel start sites and wrongly annotated pseudogenes. Most novelties were confirmed by targeted, parallel reaction monitoring mass spectrometry, including unique ORFs and single amino acid variations (SAAVs) identified in a re-sequenced laboratory strain that are not present in its reference genome. We demonstrate the general applicability of our strategy for genomes with varying GC content and distinct taxonomic origin. We release iPtgxDBs for B. henselae, Bradyrhozibium diazoefficiens and Escherichia coli and the software to generate both proteogenomics search databases and integrated annotation files that can be viewed in a genome browser for any prokaryote.

Publications that relied on an early prototype of our proteogenomics strategy

1. U. Omasits, M. Quebatte, D. J. Stekhoven, C. Fortes, B. Roschitzki, M. D. Robinson, C. Dehio, and C. H. Ahrens. 2013. Directed shotgun proteomics guided by saturated RNA-seq identifies a complete expressed prokaryotic proteome. Genome Research 23:1916-1927. 10.1101/gr.151035.112.
2. A.L. Carlier, U. Omasits, C. H. Ahrens, and L. Eberl. 2013. Proteomics analysis of Psychotria leaf nodule symbiosis: improved genome annotation and metabolic predictions. Molecular Plant-Microbe Interactions 26:1325-1333. 10.1094/MPMI-05-13-0152-R.
3. J. Čuklina, J. Hahn, M. Imakaev, U. Omasits, K. U. Förstner, N. Ljubimov, M. Goebel, G. Pessi, H. M. Fischer, C. H. Ahrens, M. S. Gelfand, and E. Evguenieva-Hackenberg. 2016. Genome-wide transcription start site mapping of Bradyrhizobium japonicum grown free-living or in symbiosis - a rich resource to identify new transcripts, proteins and to study gene regulation. BMC Genomics 17:302. 10.1186/s12864-016-2602-9.