Quantitation of Mycobacterial PDIM Lipids

Liquid chromatography-mass spectrometric quantitation of PDIM was performed on total cell wall extracts of Mtb according to published protocols [48 (link)]. In brief, Mtb strains were grown in 7H9 supplemented with Middlebrook OADC (BD) and glycerol 0.2%. Cells were then pelleted and extracted in chloroform:methanol and an equal quantity of total lipids were run through an established LC-MS protocol using an Agilent Technologies 6520 Accurate-Mass Q-Tof and a 1200 series HPLC system with a Monochrom diol column [48 (link)]. PDIM species were identified using positive mode MS based on predicted retention time [48 (link)], highly accurate mass matching to known PDIM species as listed in the figures, and confirmed based on collision-induced dissociation mass spectrometry with major fragments as listed in S1 Text.

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Barczak A.K., Avraham R., Singh S., Luo S.S., Zhang W.R., Bray M.A., Hinman A.E., Thompson M., Nietupski R.M., Golas A., Montgomery P., Fitzgerald M., Smith R.S., White D.W., Tischler A.D., Carpenter A.E, & Hung D.T. (2017). Systematic, multiparametric analysis of Mycobacterium tuberculosis intracellular infection offers insight into coordinated virulence. PLoS Pathogens, 13(5), e1006363.

Publication 2017

Middlebrook OADC 6520 Accurate-Mass Q-Tof

Cell wall Cells Chloroform Glycerol Hplc Lipids Liquid chromatography Mass spectrometry Methanol Retention Strains

Corresponding Organization : University of Minnesota

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Variable analysis

independent variables

Mtb strains

dependent variables

Quantitation of PDIM in total cell wall extracts of Mtb

control variables

Growth of Mtb strains in 7H9 supplemented with Middlebrook OADC and glycerol 0.2%
Extraction of total lipids in chloroform:methanol
LC-MS protocol using an Agilent Technologies 6520 Accurate-Mass Q-Tof and a 1200 series HPLC system with a Monochrom diol column

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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