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Mycobacterium Tuberculosis Resource Aims to Enable Proteomic Studies

NEW YORK (GenomeWeb News) – Researchers at the Swiss Federal Institute of Technology have developed a tool to single out specific proteins contained within the Mycobacterium tuberculosis proteome.

This atlas, called the Mtb Proteome Library, consists of validated selected reaction monitoring assays for nearly all M. tuberculosis proteins, as ETH-Zurich's Ruedi Aebersold and his colleagues reported today in Cell Host and Microbe.

The protein atlas includes assays for 97 percent of the more than 4,000 annotated tuberculosis proteins.

"Given access to an SRM-capable mass spectrometer, these SRM assays can be directly applied to many proteins in parallel and to any number of samples without the need to invest in the time-consuming and costly development of antibodies," Aebersold and his colleagues noted.

The researchers applied their library to study the Mtb dormancy regulon under hypoxic conditions, finding that they could quantify 45 of the 53 proteins that make up the regulon.

To develop the Mtb Proteome Library, Aebersold and his colleagues mapped the M. tuberculosis proteome by extracting and digesting protein from M. tuberculosis bacterial cultures. After fractionation and discovery mass spectrometry, the researchers assigned 144,175 fragment ion spectra to 36,924 peptides that, in turn, mapped to a little more than 3,000 of the 4,012 annotated M. tuberculosis proteins. This, they said, covered 95 percent of the MS-observable proteome, and about 86 percent of the transcriptome identified through RNA sequencing analyses.

From this, the researchers developed synthetic peptides covering the annotated M. tuberculosis proteome, totaling 17,463 peptides covering 3,930 proteins. These peptides served as sort of pre-made SRM assays that contain the most intense peptide fragment ions from the spectral libraries, the relative intensities, and the chromatographic retention time.

"Importantly, the majority of the proteins are represented by three or more peptides," the researchers noted, adding that the synthetic peptides were also analyzed on an Orbitrap and a TripleTOF mass spectrometer.

To test the utility of their library, Aebersold and his colleagues examined the protein-level regulation of the DosR regulon under hypoxic conditions in the model M. bovis strain.

They found assays for 51 of the 52 DosR regulon members in their atlas, and added the related DosT kinase to their study. Each of their target proteins, the researchers noted, were represented by three peptides with five transitions each.

Over their hypoxia time course, they were able to quantify 45 of those 53 proteins. The most highly induced protein was HspX, followed by Hrp1, they noted.

"In conclusion, we could identify and quantify almost the complete DosR regulon using the Mtb Proteome Library, thereby facilitating straightforward SRM experiments by providing high-quality and ready-to-use quantitative protein assays together with information on [the] detectability of each peptide in standard cultures," Aebersold and his colleagues wrote.

They added that the Mtb Proteome Library could serve as a resource for a number of targeted, hypothesis-driven proteomic studies.

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