Large-scale proteogenomics characterization of the <i>Mycobacterium tuberculosis</i> hidden microproteome

ABSTRACT Tuberculosis remains a burden to this day, due to the rise of multi and extensively drug-resistant bacterial strains. The genome of Mycobacterium tuberculosis (Mtb) underwent an annotation process that excluded small Open Reading Frames (smORFs), which encode a class of peptides and small proteins collectively known as microproteins. As a result, there is an overlooked part of its proteome that is a rich source of potentially essential, druggable molecular targets. Here, we employed our recently developed proteogenomics pipeline to identify novel microproteins encoded by smORFs in the genome of Mtb usings hundreds of mass spectrometry experiments in a large-scale approach. We found protein evidence for hundreds of novel microproteins and identified smORFs potentially involved in bacterial growth and virulence. Moreover, many smORFs are co-expressed or share operons with a myriad of biologically relevant genes and may play a role in antibiotic response. Together, our data presents a resource of unknown genes that play a role in the success of Mtb as a widespread pathogen.