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Fig. 3 MS analysis of crude lipid extracts from 3-azido pHB-treated M. marinum. (A) Ion chromatograms of the representative species of PGL (1523.3915 m/z) and PGL-N3 (1564.3929 m/z) detected in the total lipid extracts of M. marinum treated with 3-azido pHB (750 μM) were generated by positive-mode reversed-phase HPLC-Q-TOF-MS. (B) Mass spectra of indicated PGL and PGL-N3 species. (C) CID-MS of PGL and PGL-N3 showing diagnostic fragments corresponding to the loss of one mycocerosic acyl moiety (PGL: 1095.953 m/z; PGL-N3: 1108.948 m/z with spontaneous loss of N2 from the N3 group), the loss of one mycocerosyl moiety plus the monosaccharide (PGL: 935.879 m/z; PGL-N3: 976.880 m/z and 948.866 m/z with spontaneous loss of N2 from the N3 group), and the loss of both mycocerosic acyl moieties plus the monosaccharide (PGL: 525.467 m/z; PGL-N3: 566.468 m/z and 538.462 m/z with a spontaneous loss of N2 from the N3 group). (D) Effects of M. marinum treated with 3-azido pHB on the abundance of PGL-N3 species, as quantified by MS. The data represented as three repeated mass spectral measurements. Statistical analysis was performed using a two-way ANOVA followed by a Dunnett’s multiple comparisons test. Significance is represented by ****p < 0.0001 and ns (not significant) for p > 0.05. (E) Effects of M. marinum treated with 3-azido pHB on the abundance of PGL-N3, total PGL, PDIM, and PE as determined by MS analysis. Quantified MS data are representative of three repeated mass spectral measurements. Statistical analysis was performed using a two-way ANOVA followed by a Dunnett’s multiple comparisons test. Significance is represented as follows: *p ≤ 0.05, **p < 0.01, ****p < 0.0001, and ns (not significant) for p > 0.05.