The Mtb Proteome Library: A Resource of Assays to Quantify the Complete Proteome of Mycobacterium tuberculosis

Abstract

SUMMARY Research advancing our understanding of Mycobacterium tuberculosis (Mtb) biology and complex host-Mtb interactions requires consistent and precise quantitative measurements of Mtb proteins. We describe the generation and validation of a compendium of assays to quantify 97% of the 4,012 annotated Mtb proteins by the targeted mass spectrometric method selected reaction monitoring (SRM). Furthermore, we estimate the absolute abundance for 55% of all Mtb proteins, revealing a dynamic range within the Mtb proteome of over four orders of magnitude, and identify previously un-annotated proteins. As an example of the assay library utility, we monitored the entire Mtb dormancy survival regulon (DosR), which is linked to anaerobic survival and Mtb persistence, and show its dynamic protein-level regulation during hypoxia. In conclusion, we present a publicly available research resource that supports the sensitive, precise, and reproducible quantification of virtually any Mtb protein by a robust and widely accessible mass spectrometric method.

Disclosure of Selective Advantages in the “modern” Sublineage of the Mycobacterium tuberculosis Beijing Genotype Family by Quantitative Proteomics

“…The categories containing the PE/PPE-family and insertion sequences and phages are notoriously difficult to identify. A recent study using "proteome-wide selected reaction monitoring" also suffered from an under representation of the PE/PPE-family (39). The PE/PPE family is named after their PE proline-glutamic acid and PPE proline-proline-glutamic acid N-terminal motif.…”

“…Members of this family are characterized by a high number of glycine and alanine residues in combination with repetitive sequences. As a result, there are relatively few tryptic cleavage sites (39). This lack of tryptic digestion sites makes our workflow with endoLys-c and trypsin digestion less suitable for the detection of members from this protein family.…”

“…Efflux pumps were also identified in the proteome of M. tuberculosis H37Rv by a proteome-wide selected reaction monitoring approach (39). We compared this comprehensive dataset of 2195 M. tuberculosis H37Rv proteins with the proteins we identified.…”

“…We demonstrate the presence of multiple antibiotic extruding efflux pumps in the proteome of M. tuberculosis Beijing, notably without exposure of the pathogen to a drug. Three of these efflux pumps were previously not quantified in the proteome of M. tuberculosis H37Rv by a proteome-wide selected reaction monitoring approach (39).…”

Applications of targeted proteomics in systems biology and translational medicine

“…Following the successful quantification of single proteins or small protein lists, assays for targeted proteomics were established for entire model organisms 16, 46, 47. Applications using medium to large‐sized target lists as is the case for the study of biological networks have pushed technology development.…”