Journal: Nature chemical biology

Article Title: Mycobacterium tuberculosis releases an antacid that remodels phagosomes

doi: 10.1038/s41589-019-0336-0

Figure Lengend Snippet: a) 1-TbAd and N6-TbAd were detected as HPLC-MS ion-chromatograms. b) Raw MS counts for 1-TbAd and phosphatidylinositol (PI) were measured in 52 clinical strains and the laboratory strain H37Rv. c) Human monocytes were treated with cytokines followed by a TLR2 agonist (Pam3Cys) or 1-TbAd and subjected to flow measurement of activation markers in 3 independent experiments showing similar results. d) The Rv3377c-Rv3378c locus was transferred into M. kansasii, conferring production of a molecule with equivalent mass, retention and CIDMS fragments found in 1-TbAd. e) M. kansasii was grown in liquid media at various pH values in duplicate with low variance (coefficient of variation < 2 % for all but one measurement) in three independent experiments. f) pKa’s are derived from measurements of dimethylallyladenosine and related compounds(21, 22), which indicate that 1-TbAd is predominantly charged but in equilibrium with its uncharged conjugate base at neutral pH. Thus, 1-TbAd but not N6-TbAd exists as a strong conjugate base.

Article Snippet: Each dried fraction was re-dissolved in a mobile phase of hexane/isopropyl alcohol (70:30 v:v) and loaded to a 1200 series HPLC system using a normal phase column (Varian MonoChrom Diol: 3 μm x 150 mm x 2 mm) and analyzed by using an Agilent 6520 Accurate Mass Q-Tof mass spectrometer, based on published methods ( 8 ).

Techniques: Activation Assay, Derivative Assay

Journal: Nature chemical biology

Article Title: Mycobacterium tuberculosis releases an antacid that remodels phagosomes

doi: 10.1038/s41589-019-0336-0

Figure Lengend Snippet: a) Total lipids from three 1-TbAd+ and three 1-TbAd- strains were separated on normal phase silica TLC using chloroform/acetic acid/methanol/water mixtures in comparison to a 1-TbAd standard and a H37Rv lab strain. Lipids were subjected to charring after spraying with a phosphomolybdic acid solution. b) Labeled 1-TbAd was synthesized using 13C5 labeled adenosine whose mass spectral peaks did not overlap with those of natural 1-TbAd, allowing its use an in internal control. c) Total lipids from 5 patient-derived Mtb strains were weighed and subjected to HPLC-MS measurement in triplicate using the 13C-labeled internal standard to provide absolute mass values, which were expressed as the percentage of input lipid from 3 biologically independent samples.. d) For one representative clinical strain, lipids were extracted from the cell pellet (cellular) or conditioned media (shed) and measured as in c), expressed on a per cell basis based on colony forming unit (cfu) measurements from the input culture.

Article Snippet: Each dried fraction was re-dissolved in a mobile phase of hexane/isopropyl alcohol (70:30 v:v) and loaded to a 1200 series HPLC system using a normal phase column (Varian MonoChrom Diol: 3 μm x 150 mm x 2 mm) and analyzed by using an Agilent 6520 Accurate Mass Q-Tof mass spectrometer, based on published methods ( 8 ).

Techniques: Comparison, Labeling, Synthesized, Control, Derivative Assay