In the urine of a Niemann–Pick disease type C (NPC) patient, we have identified three characteristic intense peaks that have not been observed in the urine of a 3β-hydroxysteroid-Δ5-C27-steroid dehydrogenase deficiency patient or a healthy infant and adult. Based on accurate masses of the protonated molecules, we focused on two of them as candidate NPC diagnostic markers. Two synthesized authentic preparations agreed with the two compounds found in NPC patient urine in regard to both chromatographic behavior and accurate masses of the deprotonated molecules. Moreover, the isotopic patterns of the deprotonated molecules, twin peaks unique to the sulfur-containing compounds appearing in their second isotope positions, and accurate masses of product ions observed at m/z 97 also agreed between the target compounds and authentic preparations. We identified the two compounds as the sulfated cholesterol metabolites as 3β-sulfooxy-7β-hydroxy-5-cholen-24-oic acid and 3β-sulfooxy-7-oxo-5-cholen-24-oic acid. These two compounds represent more promising candidate diagnostic markers for NPC diagnosis than three other candidates that are multiple conjugates of cholesterol metabolites, 3β-sulfooxy-7β-N-acetylglucosaminyl-5-cholen-24-oic acid and its glycine and taurine conjugates, although we have reported an analytical method for determining the urinary levels of these compounds using liquid chromatography/electrospray ionization tandem mass spectrometry, because of their lack of N-acetylglucosamine conjugation.
Mass spectrometry imaging (MSI) with ambient sampling and ionization can rapidly and easily capture the distribution of chemical components in a solid sample. Because the spatial resolution of MSI is limited by the size of the sampling area, reducing sampling size is an important goal for high resolution MSI. Here, we report the first use of a nanopipette for sampling and ionization by tapping-mode scanning probe electrospray ionization (t-SPESI). The spot size of the sampling area of a dye molecular film on a glass substrate was decreased to 6 μm on average by using a nanopipette. On the other hand, ionization efficiency increased with decreasing solvent flow rate. Our results indicate the compatibility between a reduced sampling area and the ionization efficiency using a nanopipette. MSI of micropatterns of ink on a glass and a polymer substrate were also demonstrated.
Mycolic acids (MAs) are characteristic components of bacteria in the suborder Corynebacterineae, such as Mycobacterium. MAs are categorized into subclasses based on their functional bases (cyclopropane ring, methoxy, keto, and epoxy group). Since MAs have heterogeneity among bacterial species, analyzing of MAs are required in the chemotaxonomic field. However, their structural analysis is not easy because of their long carbon-chain lengths and several functional groups. In this study, total fatty acid (FA) methyl ester (ME) fraction of M. tuberculosis H37Rv was analyzed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOFMS) with a spiral ion trajectory (MALDI spiral-TOFMS). The distributions of carbon-chain length and their relative peak intensities were confirmed with those obtained by analysis of each subclass fraction which was separated from total FA ME fraction using thin-layer chromatography (TLC). The observed major peaks were reliably assigned as MAs owing to the high mass accuracy (error<3 ppm). The types of MA subclasses, their distributions of carbon-chain lengths, their relative peak intensities, and the ratio of even- and odd-numbered carbon-chain MAs for the total FA ME fraction were consistent with those of MA subclass fractions. To visualize whole MAs, contour maps of relative peak intensities for whole MAs were created. The contour maps indicated the MA subclasses and their distributions of carbon-chains with relative peak intensities at a glance. Our proposed method allows simple characterization in a short time and thus enables the analysis of large numbers of samples, and it would contribute to the chemotaxonomy.