Proteogenomic Analysis of Mycobacterium smegmatis Using High Resolution Mass Spectrometry

Matthys G. Potgieter, Kehilwe C. Nakedi, Jon M. Ambler, Andrew J. M. Nel, Shaun Garnett, Nelson C. Soares, Nicola Mulder, Jonathan M. Blackburn

Biochemical evidence is vital for accurate genome annotation. The integration of experimental data collected at the proteome level using high resolution mass spectrometry allows for improvements in genome annotation by providing evidence for novel gene models, while validating or modifying others. Here, we report the results of a proteogenomic analysis of a reference strain of Mycobacterium smegmatis (mc(2)155), a fast growing model organism for the pathogenic Mycobacterium tuberculosis-the causative agent for Tuberculosis. By integrating high throughput LC/MS/MS proteomic data with genomic six frame translation and ab initio gene prediction databases, a total of 2887 ORFs were identified, including 2810 ORFs annotated to a Reference protein, and 63 ORFs not previously annotated to a Reference protein. Further, the translational start site (TSS) was validated for 558 Reference proteome gene models, while upstream translational evidence was identified for 81. In addition, N-terminus derived peptide identifications allowed for downstream TSS modification of a further 24 gene models. We validated the existence of six previously described interrupted coding sequences at the peptide level, and provide evidence for four novel frameshift positions. Analysis of peptide posterior error probability (PEP) scores indicates high-confidence novel peptide identifications and shows that the genome of M. smegmatis mc(2)155 is not yet fully annotated. Data are available via ProteomeXchange with identifier PXD003500.