Yuki Sugiura

Mass Imaging and Identification of Biomolecules with MALDI-QIT-TOF-Based System

Imaging mass spectrometry is becoming a popular visualization technique in the medical and biological sciences. For its continued development, the ability to both visualize and identify molecules directly on the tissue surface using tandem mass spectrometry (MSn) is essential. We established an imaging system based on a matrix-assisted laser/desorption ionization quadrupole ion trap time-of-flight type instrument (AXIMA-QIT, Shimadzu, Kyoto, Japan), which was compatible with both imaging and highly sensitive MSn. In this paper, we present the operating conditions of the AXIMA-QIT as an imaging instrument and introduce the data converter we developed that is available free of charge. The converted data can be applied to Biomap, the commonly used visualization software. For the feasibility experiments, we demonstrated the visualization of phospholipids, glycolipid, and tryptic-digested proteins in the mouse cerebellum. The visualized lipids were successfully identified by MSn directly on the tissue surface, with a strong ability to isolate precursor ions. In the analysis of tryptic-digested proteins, we compared the product ion spectra between AXIMA-QIT and a tandem TOF-type instrument. The results confirmed that AXIMA-QIT can provide a high quality of product ion spectra even on the tissue surface.

Nanoparticle-Assisted Laser Desorption/Ionization Based Mass Imaging with Cellular Resolution

Today, two-dimensional mass spectrometry analysis of biological tissues by means of a technique called mass imaging, mass spectrometry imaging (MSI), or imaging mass spectrometry (IMS) has found application in investigating the distribution of moleculesMSI with matrix-assisted laser desorption/ionization (MALDI) and secondary ion MS (SIMS). However, the size of the matrix crystal and the migration of analytes can decrease the spatial resolution in MALDI, and SIMS can only ionize compounds with relatively low molecular weights. To overcome these problems, we developed a nanoparticle-assisted laser desorption/ionization (nano-PALDI)-based MSI. We used nano-PALDI MSI to visualize lipids and peptides at a resolution of 15 microm in mammalian tissues.

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 Visualization of Drug and Endogenous Metabolite Distribution: Toward In Situ Pharmacometabolomes

It is important to determine how a candidate drug is distributed and metabolized within the body in early phase of drug discovery. Recently, matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS; also referred to as mass spectrometry imaging) has attracted great interest for monitoring drug delivery and metabolism. Since this emerging technique enables simultaneous imaging of many types of metabolite molecules, MALDI-IMS can visualize and distinguish the parent drug and its metabolites. As another important advantage, changes in endogenous metabolites in response to drug administration can be mapped and evaluated in tissue sections. In this review, we discuss the capabilities of current IMS techniques for imaging metabolite molecules and summarize representative studies on imaging of both endogenous and exogenous metabolites. In addition, current limitations and problems with the technique are discussed, and reports of progress toward solving these problems are summarized. With this new tool, the pharmacological research community can begin to map the in situ pharmacometabolome.

Imaging Mass Spectrometry Technology and Application on Ganglioside Study; Visualization of Age-Dependent Accumulation of C20-Ganglioside Molecular Species in the Mouse Hippocampus

Gangliosides are particularly abundant in the central nervous system (CNS) and thought to play important roles in memory formation, neuritogenesis, synaptic transmission, and other neural functions. Although several molecular species of gangliosides have been characterized and their individual functions elucidated, their differential distribution in the CNS are not well understood. In particular, whether the different molecular species show different distribution patterns in the brain remains unclear. We report the distinct and characteristic distributions of ganglioside molecular species, as revealed by imaging mass spectrometry (IMS). This technique can discriminate the molecular species, raised from both oligosaccharide and ceramide structure by determining the difference of the mass-to-charge ratio, and structural analysis by tandem mass spectrometry. Gangliosides in the CNS are characterized by the structure of the long-chain base (LCB) in the ceramide moiety. The LCB of the main ganglioside species has either 18 or 20 carbons (i.e., C18- or C20-sphingosine); we found that these 2 types of gangliosides are differentially distributed in the mouse brain. While the C18-species was widely distributed throughout the frontal brain, the C20-species selectively localized along the entorhinal-hippocampus projections, especially in the molecular layer (ML) of the dentate gyrus (DG). We revealed development- and aging-related accumulation of the C-20 species in the ML-DG. Thus it is possible to consider that this brain-region specific regulation of LCB chain length is particularly important for the distinct function in cells of CNS.

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.

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.

In situ proteomics with imaging mass spectrometry and principal component analysis in the Scrapper-knockout mouse brain

Imaging MS is emerging as a useful tool for proteomic analysis. We utilized this technique to analyze gene knockout (KO) mice in addition to traditional 2‐DE analysis. The Scrapper‐knockout (SCR‐KO) mouse brain showed two types of neurodegenerative pathologies, the spongiform neurodegeneration and shrinkage of neuronal cells. 2‐DE analysis of the whole brain lysates of SCR‐KO mice indicated slight changes in annexin A6, Rap1 GTPase, and glyoxalase domain containing four spots while most of the main components did not show significant changes. By imaging MS analysis based on principal component analysis (PCA), we could find numerous alterations in the KO mouse brain. Furthermore, we could also know the information on the position of altered substances all together. PCA provides information about which molecules in tissue microdomains have altered and is helpful in analyzing large dataset of imaging MS, while exact identification of each molecule from peaks in MALDI imaging MS may require additional analyses such as MS/MS. Direct imaging with PCA is a powerful tool to perform in situ proteomics and will lead to novel findings. Our study shows that imaging MS yields information complementary to conventional 2‐DE analysis.

Selective imaging of positively charged polar and nonpolar lipids by optimizing matrix solution composition

Previous studies have shown that matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) is useful for studying the distribution of various small metabolites, particularly lipids. However, in this technique, selective ionization of the target molecules is imperative, particularly when analyzing small molecules. Since the sample clean-up procedures available for the MALDI-IMS of small metabolites are limited, the tissue sample will contain numerous molecular species other than the target molecules. These molecules will compete for ionization resulting in severe ion suppression. Hence, it is necessary to develop and optimize a sample preparation protocol for the target molecules. In this study, through model experiments using reference compounds, we optimized the composition of the matrix solution used for positively charged lipids in terms of the concentration of the organic solvent and presence/absence of alkali metal salts. We demonstrated that a high concentration of organic solvent in the matrix solution favors the preferential detection of lipids over peptides. The presence of alkali metal salts in the matrix solution was favorable for the detection of polar lipids, while a salt-free matrix solution was suitable for the detection of nonpolar lipids. Furthermore, potassium salts added to the matrix solution caused merging of various lipid adducts (adducts with proton, sodium, and potassium) into one single potassiated species. Using the optimized protocols, we selectively analyzed phosphatidylcholine (PC) and triacylglycerol (TG) with different fatty acid compositions in a rat kidney section.

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).