Dataset Information

Reanalysis of Data-dependent acquisition based proteomic datasets of Mycobacterium smegmatis

ABSTRACT: Detection of species-specific proteotypic peptides for accurate and easy characterization of infectious non-tuberculous mycobacteria such as Mycobacterium smegmatis is essential. Therefore, we carried out reanalysis of publicly available M. smegmatis proteomic dataset PXD003303, PXD004197, PXD010020, PXD016241, PXD017602, PXD027848, PXD027855 and PXD029588 by proteome database search and followed by spectral library generation. The raw DDA data were searched against their respective reference proteome databases using Proteome Discoverer and FragPipe. The resulting peptide spectrum matches were converted into a spectral library using BiblioSpec.

INSTRUMENT(S): LTQ Orbitrap Velos, Q Exactive HF, Q Exactive

ORGANISM(S): Mycobacterium Smegmatis

SUBMITTER: Keshava Prasad T. S.

LAB HEAD: Dr T.

PROVIDER: PXD046063 | Pride | 2024-03-07

REPOSITORIES: Pride

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Publications

Discovery of Species-Specific Proteotypic Peptides To Establish a Spectral Library Platform for Identification of Nontuberculosis Mycobacteria from Mass Spectrometry-Based Proteomics.

Kotimoole Chinmaya Narayana CN Ramya Vadageri Krishnamurthy VK Kaur Parvinder P Reiling Norbert N Shandil Radha Krishan RK Narayanan Shridhar S Flo Trude Helen TH Prasad Thottethodi Subrahmanya Keshava TSK

Journal of proteome research 20240215 3

Nontuberculous mycobacteria are opportunistic bacteria pulmonary and extra-pulmonary infections in humans that closely resemble <i>Mycobacterium tuberculosis</i>. Although genome sequencing strategies helped determine NTMs, a common assay for the detection of coinfection by multiple NTMs with <i>M. tuberculosis</i> in the primary attempt of diagnosis is still elusive. Such a lack of efficiency leads to delayed therapy, an inappropriate choice of drugs, drug resistance, disease complications, mor ...[more]

PMID: 38358903

Publication: 1/8

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Project description:CarD is an essential mycobacterial protein that we had previously shown to bind the RNA polymerase (RNAP) and affect the transcriptional profile of M. smegmatis and Mycobacterium tuberculosis. For this reason, we suspected that CarD was directly regulating transcriptional complexes but we did not know at what stage of CarD was functioning and at which genes CarD interacted with the RNAP. To determine in which stage of the transcription cycle (initiation, elongation, or termination) CarD acts, we used Chromatin Immunoprecipitation sequencing (ChIP-seq) to survey the distribution of CarD throughout the M. smegmatis chromosome. Specific antibodies targeting core RNAPb, RNAPσ, or a hemagglutinin (HA) epitope fused to CarD (CarD-HA) were used to co-immunoprecipitate associated DNA. CarD-HA was immunoprecipitated from the M. smegmatis Mc2155 ΔcarD attB::tetcarD-HA strain and unfused HA was immunoprecipitated from the Mc2155 attB::pmsg431 strain with monoclonal antibodies specific for HA (Sigma). RNAP β and σ were immunoprecipitated from M. smegmatis ΔcarD attB::tetcarD-HA with monoclonal antibodies specific for these subunits (Neoclone, Madison, WI; 8RB13 for β, 2G10 for σ). Co-precipitated DNA was sequenced using a SOLiD sequencer (Life Technologies), which provided sufficient reads for 100-fold coverage of the genome. The number of sequence reads per base pair was normalized to the total number of reads and expressed as a log2 value. The reads per base pair from the HA-alone sample served as the background and was subtracted from the other datasets. We found that CarD was never present on the genome in the absence of RNAP. However, whereas RNAP core enzyme was found throughout transcribed regions of the genome, CarD was primarily associated with promoter regions and highly correlated with RNAPσ. The colocalization of σA and CarD led us to propose that in vivo, CarD associates with RNAP initiation complexes at most promoters and is therefore a global regulator of transcription initiation. The genome sequences associated with M. smegmatis CarD, RNAPb, and RNAPs were determined by ChIP-seq analysis. Samples were done in duplicate, except for RNAPs. And sequencing was performed using a SOLiD sequencer (Life Technologies).

Dataset Information

Reanalysis of Data-dependent acquisition based proteomic datasets of Mycobacterium smegmatis

Publications

Discovery of Species-Specific Proteotypic Peptides To Establish a Spectral Library Platform for Identification of Nontuberculosis Mycobacteria from Mass Spectrometry-Based Proteomics.

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