The stratum corneum (SC), the outermost barrier of mammalian bodies, consists of layers of cornified keratinocytes with intercellular spaces sealed with lipids. The insolubility of the SC has hampered in-depth analysis, and the SC has been considered a homogeneous barrier. Here, we applied time-of-flight secondary ion mass spectrometry to demonstrate that the SC consists of three layers with distinct properties. Arginine, a major component of filaggrin-derived natural moisturizing factors, was concentrated in the middle layer, suggesting that this layer functions in skin hydration. Topical application of metal ions revealed that the outer layer allowed their passive influx and efflux, while the middle and lower layers exhibited distinct barrier properties, depending on the metal tested. Notably, filaggrin deficiency abrogated the lower layer barrier, allowing specific metal ions to permeate viable layers. These findings elucidate the multi-layered barrier function of the SC and its defects in filaggrin-deficient atopic disease patients.
Neurons have a large surface because of their long and thin neurites. This surface is composed of a lipid bilayer. Lipids have not been actively investigated so far because of some technical difficulties, although evidence from cell biology is emerging that lipids contain valuable information about their roles in the central nervous system. Recent progress in techniques, e.g., mass spectrometry, opens a new epoch of lipid research. We show herein the characteristic localization of phospholipid components in neurites by means of time-of-flight secondary ion mass spectrometry. We used explant cultures of mouse superior cervical ganglia, which are widely used by neurite investigation research. In a positive-ion detection mode, phospholipid head group molecules were predominantly detected. The ions of m/z 206.1 [phosphocholine, a common component of phosphatidylcholine (PC) and sphingomyelin (SM)] were evenly distributed throughout the neurites, whereas the ions of m/z 224.1, 246.1 (glycerophosphocholine, a part of PC, but not SM) showed relatively strong intensity on neurites adjacent to soma. In a negative-ion detection mode, fatty acids such as oleic and palmitic acids were mainly detected, showing high intensity on neurites adjacent to soma. Our results suggest that lipid components on the neuritic surface show characteristic distributions depending on neurite region.
Imaging mass spectrometry (IMS) provides a novel opportunity for visualization of molecular ion distribution. Currently, there are two major ionization techniques, matrix-assisted laser desorption/ionization (MALDI) and secondary ion mass spectrometry (SIMS) are widely used for imaging of biomolecules in tissue samples. MALDI and SIMS-based IMS have the following features; measurable mass ranges are wide and small, and the spatial resolutions are low and high, respectively. To the best of our knowledge, this is a first report to identify the lipids in cultured mammalian neurons by MALDI-IMS. Further, those neurons were analyzed with SIMS-IMS in order to compare the distribution pattern of lipids and other derived fragments. The parameters which influence the identification of lipids in cultured neurons were optimized in order to get an optimum detection of lipid molecules. The combined spatial data of MALDI and SIMS supported the idea that the signals of small molecules such as phosphatidylcholine head groups and fatty acids (detected in SIMS) are derived from the intact lipids (detected in MALDI-IMS).
and Mitsutoshi Setou a * Breast cancer is the most common cancer among women worldwide. The molecular characterization of breast tumor cells by using single-cell lipidomics remains relatively unexplored. Here, we introduce a time-of-flight secondary-ion mass spectrometry (TOF-SIMS) approach to visualize the lipids in individual breast cancer cells. The SKBR-3 breast cancer cell line was cultured and dispersed into individual cells. After attachment to a substrate, the cells were rinsed with ammonium acetate and were analyzed using TOF-SIMS. The instrument was operated with Bi 3 2+ as the primary ion. The distributions of ions, including positively charged phosphocholine, and negatively charged phosphates and fatty acids, were simultaneously visualized. These ions were distributed predominantly at the cell attachment sites. The signal intensities of fatty acid ions were determined from the mass spectra at the regions-of-interest. The results of fatty acid analyses on breast cancer cells were consistent with those of our previous study in which prominent expression of stearoyl-CoA desaturase 1 in breast cancer cells was demonstrated. Static TOF-SIMS was shown to be an effective method for determining the lipid molecular signature of the plasma membrane of individual breast cancer cells.
The paramagnetic metal chelate complex Cu(2+)-iminodiacetic acid (Cu(2+)-IDA) was mixed with ubiquitin, a small globular protein. Quantitative analyses of (1)H and (15)N chemical shift changes and line broadenings induced by the paramagnetic effects indicated that Cu(2+)-IDA was localized to a histidine residue (His68) on the ubiquitin surface. The distances between the backbone amide proton and the Cu(2+) relaxation center were evaluated from the proton transverse relaxation rates enhanced by the paramagnetic effect. These correlated well with the distances calculated from the crystal structure up to 20 A. Here, we show that a Cu(2+)-IDA is the first paramagnetic reagent that specifically localizes to a histidine residue on the protein surface and gives the long-range distance information.
Lipid metabolism has attracted much attention in tumor biology studies. Recently, time-of-flight secondary ion-mass spectrometry (TOF-SIMS) imaging has enabled in situ lipid analysis of biological specimens with a submicrometer spatial resolution for analyses of tumor cells. In clinical settings, sample preservation is important, and specimens are often obtained from patients at different times; these samples must be preserved prior to measurement, and preservation techniques commonly involve chemical fixation with aldehydes. However, the influence of sample preservation on TOF-SIMS analysis of fatty acids has not been reported. Thus, we examined the influence of glutaraldehyde fixation on TOF-SIMS analyses by using the multiple myeloma cell line U266. We prepared two indium-tin-oxide-coated glass slides on which cells were attached. One slide was fixed with 0.25% glutaraldehyde and rinsed in ammonium acetate buffer, whereas the other was left untreated. The specimens were subjected to TOF-SIMS analyses in negative-ion mode, and signals in the mass range of m/z 0-1850 were monitored. Both fixed and unfixed cells exhibited intense ion peaks corresponding to phosphoric acids and five types of fatty acids, putatively derived from membrane phospholipids. These ions were localized at the cell attachment site. We statistically compared the mean intensity of fatty acids between fixed and unfixed cells and found that both showed equivalent signals. Glutaraldehyde fixation was thus shown to be an effective method for preparing samples for single-cell lipid analysis by TOF-SIMS.
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