Naoko Goto-Inoue

Matrix‐assisted laser desorption/ionization quadrupole ion trap time‐of‐flight (MALDI‐QIT‐TOF)‐based imaging mass spectrometry reveals a layered distribution of phospholipid molecular species in the mouse retina

We recently developed a matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight (MALDI-QIT-TOF)-based imaging mass spectrometry (IMS) system. This system enables us to perform structural analyses using tandem mass spectrometry (MS/MS), as well as to visualize phospholipids and peptides in frozen sections. In the retina, phototransduction is regulated by the light-sensitive interaction between visual pigment-coupled receptor proteins, such as rhodopsin, and G proteins, such as transducin. There are some reports that the conformation of rhodopsin is influenced by the composition of phospholipids in the lipid bilayer membrane. However, these results were based on in vitro experiments and have not been analyzed in vivo. In this study, we visualized and identified phospholipids in mouse retinal sections with the MALDI-QIT-TOF-based IMS system. From a spectrum obtained by raster-scanned analysis of the sections, ions with high signal intensities were selected and analyzed by MS/MS. As a result, sixteen ions were identified as being from four diacyl-phosphatidylcholine (PC) species, i.e., PC (16:0/16:0), PC (16:0/18:1), PC (16:0/22:6), and PC (18:0/22:6), with different ion forms. The ion images revealed different distributions on the retinal sections: PC (16:0/18:1) was distributed in the inner nuclear layer and outer plexiform layer, PC (16:0/16:0) in the outer nuclear layer and inner segment, and both PC (16:0/22:6) and PC (18:0/22:6) in the outer segment and pigment epithelium. In conclusion, our in vivo IMS analyses demonstrated a three-zone distribution of PC species on the retinal sections. This approach may be useful for analyzing lipid changes and their contribution to phototransduction in the retina.

Imaging mass spectrometry for lipidomics.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful tool that enables the simultaneous detection and identification of biomolecules in analytes. MALDI-imaging mass spectrometry (MALDI-IMS) is a two-dimensional MALDI-MS technique used to visualize the spatial distribution of biomolecules without extraction, purification, separation, or labeling of biological samples. This technique can reveal the distribution of hundreds of ion signals in a single measurement and also helps in understanding the cellular profile of the biological system. MALDI-IMS has already revealed the characteristic distribution of several kinds of lipids in various tissues. The versatility of MALDI-IMS has opened a new frontier in several fields, especially in lipidomics. In this review, we describe the methodology and applications of MALDI-IMS to biological samples.

Visualization of volatile substances in different organelles with an atmospheric-pressure mass microscope

We have developed a mass microscope (mass spectrometry imager with spatial resolution higher than the naked eye) equipped with an atmospheric pressure ion-source chamber for laser desorption/ionization (AP-LDI) and a quadrupole ion trap time-of-flight (QIT-TOF) analyzer. The optical microscope combined with the mass spectrometer permitted us to precisely determine the relevant tissue region prior to performing imaging mass spectrometry (IMS). An ultraviolet laser tightly focused with a triplet lens was used to achieve high spatial resolution. An atmospheric pressure ion-source chamber enables us to analyze fresh samples with minimal loss of intrinsic water or volatile compounds. Mass-microscopic AP-LDI imaging of freshly cut ginger rhizome sections revealed that 6-gingerol ([M + K](+)at m/z 333.15, positive mode; [M - H](-) at m/z 293.17, negative mode) and the monoterpene ([M + K](+) at m/z 191.09), which are the compounds related to pungency and flavor, respectively, were localized in oil drop-containing organelles. AP-LDI-tandem MS/MS analyses were applied to compare authentic signals from freshly cut ginger directly with the standard reagent. Thus, our atmosphere-imaging mass spectrometer enabled us to monitor a quality of plants at the organelle level.

Application of imaging mass spectrometry for the analysis of Oryza sativa rice

Rice is one of the most important food crops in the world and new varieties have been bred for specific purposes, such as the development of drought-resistance, or the enrichment of functional food factors. The localization and composition of metabolites in such new varieties must be investigated because all artificial interventions are expected to change the metabolites of rice. Imaging mass spectrometry using matrix-assisted laser desorption/ionization (MALDI-IMS) is a suitable tool for investigating the localization and composition of metabolites; however, suitable methodologies for the MALDI-IMS analysis of rice have not yet been established. In this study, we optimized the methods for analyzing rice grains by MALDI-IMS using adhesive film and found the characteristic distribution of metabolites in rice. Lysophosphatidylcholine (LPC) was localized in the endosperm. Phosphatidylcholine (PC), gamma-oryzanol and phytic acid were localized in the bran (germ and seed coat), and alpha-tocopherol was distributed in the germ (especially in the scutellum). In addition, MALDI-IMS revealed the LPC and PC composition of the rice samples. The LPC composition, LPC (1-acyl 16:0), LPC (1-acyl 18:2), LPC (1-acyl 18:1) and LPC (1-acyl 18:0), was 59.4 +/- 4.5%, 19.6 +/- 2.5%, 14.2 +/- 4.5% and 6.8 +/- 1.4%. The PC composition, PC (diacyl 16:0/18:2), PC (diacyl 16:0/18:1), PC (diacyl 18:1/18:3), PC (diacyl 18:1/18:2) and PC (diacyl 18:1/18:2), was 19.6 +/- 1.0%, 21.0 +/- 1.0%, 15.0 +/- 1.4%, 26.7 +/- 0.7% and 17.8 +/- 1.9%. This approach can be applied to the assessment of metabolites not only in rice, but also in other foods for which the preparation of sections is a challenging task.

Imaging mass spectrometry of gastric carcinoma in formalin‐fixed paraffin‐embedded tissue microarray

The popularity of imaging mass spectrometry (IMS) of tissue samples, which enables the direct scanning of tissue sections within a short time‐period, has been considerably increasing in cancer proteomics. Most pathological specimens stored in medical institutes are formalin‐fixed; thus, they had been regarded to be unsuitable for proteomic analyses, including IMS, until recently. Here, we report an easy‐to‐use screening method that enables the analysis of multiple samples in one experiment without extractions and purifications of proteins. We scanned, with an IMS technique, a tissue microarray (TMA) of formalin‐fixed paraffin‐embedded (FFPE) specimens. We detected a large amount of signals from trypsin‐treated FFPE‐TMA samples of gastric carcinoma tissues of different histological types. Of the signals detected, 54 were classified as signals specific to cancer with statistically significant differences between adenocarcinomas and normal tissues. We detected a total of 14 of the 54 signals as histological type‐specific with the support of statistical analyses. Tandem MS revealed that a signal specific to poorly differentiated cancer tissue corresponded to histone H4. Finally, we verified the IMS‐based finding by immunohistochemical analysis of more than 300 specimens spotted on TMAs; the immunoreactivity of histone H4 was remarkably strong in poorly differentiated cancer tissues. Thus, the application of IMS to FFPE‐TMA can enable high‐throughput analysis in cancer proteomics to aid in the understanding of molecular mechanisms underlying carcinogenesis, invasiveness, metastasis, and prognosis. Further, results obtained from the IMS of FFPE‐TMA can be readily confirmed by commonly used immunohistochemical analyses. (Cancer Sci 2009)

Imaging Mass Spectrometry with Silver Nanoparticles Reveals the Distribution of Fatty Acids in Mouse Retinal Sections

A new approach to the visualization of fatty acids in mouse liver and retinal samples has been developed using silver nanoparticles (AgNPs) in nanoparticle-assisted laser desorption/ ionization imaging mass spectrometry (nano-PALDI-IMS) in negative ion mode. So far, IMS analysis has concentrated on main cell components, such as cell membrane phospholipids and cytoskeletal peptides. AgNPs modified with alkylcarboxylate and alkylamine were used for nano-PALDI-IMS to identify fatty acids, such as stearic, oleic, linoleic, arachidonic, and eicosapentaenoic acids, as well as palmitic acid, in mouse liver sections; these fatty acids are not detected using 2,5-dihydroxybenzoic acid (DHB) as a matrix. The limit of detection for the determination of palmitic acid was 50 pmol using nano-PALDI-IMS. The nano-PALDI-IMS method is successfully applied to the reconstruction of the ion images of fatty acids in mouse liver sections. We verified the detection of fatty acids in liver tissue sections of mice by analyzing standard lipid samples, which showed that fatty acids were from free fatty acids and dissociated fatty acids from lipids when irradiated with a laser. Additionally, we applied the proposed method to the identification of fatty acids in mouse retinal tissue sections, which enabled us to learn the six-zonal distribution of fatty acids in different layers of the retina. We believe that the current approach using AgNPs in nano-PALDI-IMS could lead to a new strategy to analyze basic biological mechanisms and several diseases through the distribution of fatty acids.

Imaging mass spectrometry revealed the production of lyso-phosphatidylcholine in the injured ischemic rat brain

To develop an effective neuroprotective strategy against ischemic injury, it is important to identify the key molecules involved in the progression of injury. Direct molecular analysis of tissue using mass spectrometry (MS) is a subject of much interest in the field of metabolomics. Most notably, imaging mass spectrometry (IMS) allows visualization of molecular distributions on the tissue surface. To understand lipid dynamics during ischemic injury, we performed IMS analysis on rat brain tissue sections with focal cerebral ischemia. Sprague-Dawley rats were sacrificed at 24 h after middle cerebral artery occlusion, and brain sections were prepared. IMS analyses were conducted using matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS) in positive ion mode. To determine the molecular structures, the detected ions were subjected to tandem MS. The intensity counts of the ion signals of m/z 798.5 and m/z 760.5 that are revealed to be a phosphatidylcholine, PC (16:0/18:1) are reduced in the area of focal cerebral ischemia as compared to the normal cerebral area. In contrast, the signal of m/z 496.3, identified as a lyso-phosphatidylcholine, LPC (16:0), was clearly increased in the area of focal cerebral ischemia. In IMS analyses, changes of PC (16:0/18:1) and LPC (16:0) are observed beyond the border of the injured area. Together with previous reports--that PCs are hydrolyzed by phospholipase A(2) (PLA(2)) and produce LPCs,--our present results suggest that LPC (16:0) is generated during the injury process after cerebral ischemia, presumably via PLA(2) activation, and that PC (16:0/18:1) is one of its precursor molecules.

The specific localization of seminolipid molecular species on mouse testis during testicular maturation revealed by imaging mass spectrometry.

More than 90% of the glycolipid in mammalian testis consists of a unique sulfated glyceroglycolipid called seminolipid. The galactosylation of the molecule is catalyzed by UDP-galactose:ceramide galactosyltransferase (CGT). Disruption of the CGT gene in mice results in male infertility due to the arrest of spermatogenesis, indicating that seminolipid plays an important role in reproductive function. Seminolipid molecules can be assigned to different molecular species based on the fatty acid composition. In this report, we investigated the localizations of the molecular species of seminolipid by imaging mass spectrometry and demonstrated that major molecule (C16:0-alkyl-C16:0-acyl) was expressed throughout the tubules: some (C16:0-alkyl-C14:0-acyl and C14:0-alkyl-C16:0-acyl) were predominantly expressed in spermatocytes and the other (C17:0-alkyl-C16:0-acyl) was specifically expressed in spermatids and spermatozoa. This is the first report to show the cell-specific localization of each molecular species of seminolipid during testicular maturation.

Ionic matrix for enhanced MALDI imaging mass spectrometry for identification of phospholipids in mouse liver and cerebellum tissue sections.

The ionic matrix (IM) is considered to be versatile for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the identification of a wide range of biomolecules due to its good solubility for a variety of analytes, formation of homogeneous crystals with analytes, and high vacuum stability. When these advantages are exploited, the performance of IM of α-cyano-4-hydroxycinnamic acid butylamine (CHCAB) and 2,5-dihydroxybenzoic acid butylamine (DHBB) was compared with other matrixes for the identification of phospholipids in standard mixtures and mouse liver tissue sections. The results showed that the IM of CHCAB caused higher signal intensity and allowed the detection of a number phospholipids such as phosphatidylethanolamine (PE) and phosphatidylserine (PS) in addition to detection of phosphatidylcholine (PC) on the surface of the liver tissue sample. The IM of CHCAB was also used to identify the species of lipids present in different layers of cerebellum where the greater numbers of biomolecules were detected as compared to DHB matrix. Further, the feasibility of the proposed method was extended for the analysis of tryptic digested cytochrome c for increased signal intensity and number of peptide sequences in MALDI-MS. Thus, the application of IM to MALDI-MS could be a promising tool for imaging biomolecules in tissue sections in high throughput analyses with high sensitivity.

High-sensitivity analysis of glycosphingolipids by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight imaging mass spectrometry on transfer membranes

Glycosphingolipids are ubiquitous constituents of cells. Yet there is still room for improvement in the techniques for analyzing glycosphingolipids. Here we report our highly sensitive and convenient analytical technology with imaging mass spectrometry for detailed structural analysis of glycosphingolipids. We were able to determine detailed ceramide structures; i.e., both the sphingosine base and fatty acid, by MS/MS/MS analysis on a PVDF membrane with 10 pmol of GM1, with which only faint bands were visible by primuline staining. The limit of detection was approximately 1 pmol of GM1, which is lower than the value in the conventional reports (10 pmol).