Yasuhide Naito

Analysis of N-glycans of pathological tau: possible occurrence of aberrant processing of tau in Alzheimer's disease.

In a previous study [Wang et al. (1996) Nat. Med. 2, 871–875], Wang et al. found (i) that abnormally hyperphosphorylated tau (AD P‐tau) isolated from Alzheimers disease (AD) brain as paired helical filaments (PHF)‐tau and as cytosolic AD P‐tau but not tau from normal brain were stained by lectins, and (ii) that on in vitro deglycosylation the PHF untwisted into sheets of thin straight filaments, suggesting that tau only in AD brains is glycosylated. To elucidate the primary structure of N‐glycans, we comparatively analyzed the N‐glycan structures obtained from PHF‐tau and AD P‐tau. More than half of N‐glycans found in PHF‐tau and AD P‐tau were different. High mannose‐type sugar chains and truncated N‐glycans were found in both taus in addition to a small amount of sialylated bi‐ and triantennary sugar chains. More truncated glycans were richer in PHF‐tau than AD P‐tau. This enrichment of more truncated glycans in PHF might be involved in promoting the assembly and or stabilizing the pathological fibrils in AD.

Quantitative capability of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for a complex mixture

Electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) has been evaluated for the quantitation of complex mixtures, by using a crude oil (Taching vacuum residue) and polyethylene glycol (PEG600) as model analytes. The crude oil is an extremely complex mixture consisting of hundreds of organic compounds, which are mostly non-polar hydrocarbons. The crude oil ESI FT-ICR mass spectrum contained approximately five hundred distinguishable peaks accompanied by plenty of isobars. These ionic species were singly charged but not protonated. Although the crude oil response line was saturated at high concentration, the linearly responding range was extended by shortening the duration of the external accumulation which was carried out in the hexapole ion guide. No improvement in the linear response was observed for the exclusively trapped crude oil ions in a narrow m/z range which was trimmed by a correlated sweep ejection event. The crude oil mass distribution pattern shifted toward higher m/z as the analyte concentration became higher. A linear relationship was observed between the analyte concentrations and the average molecular weights, in both the crude oil and PEG600 experiments. The average molecular weights increased on extending the external accumulation time. This phenomenon was attributable to a novel space charge effect taking place for externally accumulated ions. Optimizing the external accumulation period was vital to get a linear response up to a reasonably high concentration region. Basically, complexity and plurality of mixture samples need not significantly affect quantitation by means of ESI FT-ICR MS.

Development of a stigmatic mass microscope using laser desorption/ionization and a multi-turn time-of-flight mass spectrometer

A novel stigmatic mass microscope using laser desorption∕ionization and a multi-turn time-of-flight mass spectrometer, MULTUM-IMG, has been developed. Stigmatic ion images of crystal violet masked by a fine square mesh grid with a 12.7 μm pitch as well as microdot patterns with a 5 μm dot diameter and a 10 μm pitch made with rhodamine B were clearly observed. The estimated spatial resolution was about 3 μm in the linear mode with a 20-fold ion optical magnification. Separating stigmatic ion images according to the time-of-flight, i.e., the mass-to-charge ratio of the ions was successfully demonstrated by a microdot pattern made with two different dyes, crystal violet and methylene blue. Stigmatic ion images of a microdot pattern made with crystal violet were observed after circulation in MULTUM-IMG, and the pattern of the ion image was maintained after ten cycles in MULTUM-IMG. A section of a mouse brain stained with crystal violet and methylene blue was observed in the linear mode, and the stigmatic total ion image of crystal violet and methylene blue agreed well with the optical microphotograph of the hippocampus for the same section.

Fragmentation study of peptides using Fourier transform ion cyclotron resonance with infrared multiphoton dissociation: experiment and simulation.

In this study, the fragmentation of gas-phase protonated Angiotensin II is investigated using electrospray ionization (ESI), Fourier transform ion cyclotron resonance (FT-ICR) and mass spectrometry (MS) with a laser cleavage infrared multiphoton dissociation (IRMPD) technique. The experimental results show that the spectra peaks for the photoproducts are y2/b6- and y7-type ions, corresponding to the cleavage of His–Pro and Asp–Arg in the parent amino acid sequence. The fragmentation of the peptide under collision-free vacuum conditions is modeled using molecular dynamics simulations (MD). The binding energy for the peptide bonds (C′–N bond) of Angiotensin II is estimated from ab initio calculations. The calculations are directed at predicting experimental measurements of the product ions from the photodissociation of the peptide. The product distributions simulated by the MD dissociation trajectories include predominantly y7/b1 and y2/b6 pair ions.

Comparison of mass spectra of peptides in different matrices using matrix‐assisted laser desorption/ionization and a multi‐turn time‐of‐flight mass spectrometer, MULTUM‐IMG

The mass spectra of peptides obtained with different matrices were compared using a matrix-assisted laser desorption/ionization (MALDI) ion source and a multi-turn time-of-flight (TOF) mass spectrometer, MULTUM-IMG, which has been developed at Osaka University. Two types of solid matrices, alpha-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB), and a liquid matrix made from a mixture of 3-aminoquinoline and CHCA were used. When measuring the peak signal intensity of human angiotensin II [M+H]+ from a fixed sample position, the liquid matrix produced a stable signal over 1000 laser shots, while the signal obtained with CHCA and DHB decayed after about 300 and 100 shots, respectively. Significant differences in the mass resolving power were not observed between the spectra obtained with the three matrices. Signal peak areas were measured as a function of the cycle number in a multi-turn ion trajectory, i.e., the total flight time over a millisecond time scale. For both [M+H]+ of human angiotensin II and bovine insulin, the decay of the signal peak area was the most significant with CHCA, while that measured with DHB was the smallest. The results of the mean initial ion velocity measurements suggested that the extent of metastable decomposition of the analyte ions increased in order of DHB, the liquid matrix, and CHCA, which is consistent with the difference in the decay of the signal peak area as the total flight time increased.

Evaluation of a Delay-Line Detector Combined with Analog-to-Digital Converters as the Ion Detection System for Stigmatic Imaging Mass Spectrometry

In addition to identifying analytes from the time-of-flight of ions, stigmatic imaging mass spectrometry (IMS) realizes microscopic and high-throughput imaging of multiple mass-resolved molecules. However, practical applications require a fast position- and time-sensitive ion detector. We have developed a delay-line detector combined with analog-to-digital converters (ADCs), and evaluated its performance as an ion detector for stigmatic IMS using electron ionization. The system shows spatial and temporal resolutions of 230 µm and 24 ns (4.2 ×107 frames per second), respectively. We speculate that the timing jitter of the signals limits the spatial resolution due to the signal processing devices. At a 20-fold ion optical magnification and m/z of 10,000, the estimated spatial resolution and mass resolving power m/Δm using this ion detection system with a stigmatic imaging mass spectrometer, which we are currently developing, are 12 µm and 15,000, respectively.

Development of a novel stigmatic imaging mass spectrometer using laser ionization and a multi-turn time-of-flight mass spectrometer

A stigmatic and microscopic imaging mass spectrometer has been developed using matrix-assisted laser desorption/ionization and a multi-turn time-of-flight mass spectrometer, MULTUM-IMG. Ion images of rhodamine 6G masked by fine grids with pitches of 63.5, 25.4, and 12.7 μm were clearly observed in the linear mode. Separation of stigmatic ion images according to the time-of-flight, i.e., the mass-to-charge ratio of ions was also successfully demonstrated with a micro-dot pattern made with crystal violet and methylene blue. Stigmatic ion images of a micro-dot pattern made with crystal violet was also observed and the ion image of the pattern was maintained after 10 cycles in MULTUM-IMG. A section of a mouse brain stained with crystal violet and methylene blue was observed in the linear mode, and the stigmatic total ion image of crystal violet and methylene blue was in good agreement with the optical microphotograph of the hippocampus in the section.

A novel laser desorption/ionization method using through hole porous alumina membranes

Rationale A novel matrix‐free laser desorption/ionization method based on porous alumina membranes was developed. The porous alumina membranes have a two‐dimensional (2D) ordered structure consisting of closely aligned straight through holes of sub‐micron in diameter that are amenable to mass production by industrial fabrication processes. Methods Considering a balance between the ion generating efficiency and the mechanical strength of the membranes, the typical values for the hole diameter, open aperture ratio and membrane thickness were set to 200 nm, 50% and 5 μm, respectively. The membranes were coated with platinum on a single side that was exposed to the laser. Evaluation experiments were conducted on the feasibility of this membrane structure for an ionization method using a single peptide and mixed peptides and polyethylene glycol samples and a commercial matrix‐assisted laser desorption/ionization (MALDI) time‐of‐flight mass spectrometer in the positive ion mode. Results Results showed a softness of ionization and no sweet spot nature. The capillary action of the through holes with very high aspect ratio enables several loading protocols including sample impregnation from the surface opposite to the laser exposure side. Conclusions The feasibility study indicates that the through hole porous alumina membranes have several advantages in terms of usefulness over the conventional surface‐assisted laser desorption ionization (SALDI) methods. The proposed novel ionization method is termed Desorption Ionization Using Through Hole Alumina Membrane (DIUTHAME).

Tissue imaging with a stigmatic mass microscope using laser desorption/ionization

A novel stigmatic mass microscope using laser desorption/ionization and a multi-turn time-of-flight mass spectrometer, MULTUM-IMG, has been developed. Stigmatic ion images of crystal violet masked by a fine square mesh grid with a 12.7 μm pitch were clearly observed, and the estimated spatial resolution was about 3 μm in the linear mode with a 20-fold ion optical magnification. Tissue sections of a brain and eyes of a mouse stained with crystal violet and methylene blue were observed in the linear mode, and the stigmatic total ion images of crystal violet and methylene blue agreed well with the optical photomicrograph of the same sections. Especially, the fine structure in the cornea tissue was clearly observed with a spatial resolution in the range of micrometers. Although the total measurement time of the stigmatic ion image for the whole-eye section was about 59 minutes using a laser with a 10 Hz repetition rate, the measurement time could be reduced to about 35 s using a laser with a 1 kHz repetition rate and automation of measurements. The stigmatic mass microscope developed in this research should be suitable for high-spatial resolution and high-throughput imaging mass spectrometry for pathology, pharmacokinetics, and so on.

Development of a stigmatic imaging mass spectrometer using laser desorption/ionization

Imaging mass spectrometry based on matrix-assisted laser desorption/ionization gives distributions of multiple biomolecules and identification of analytes in a tissue section. We are developing a stigmatic imaging mass spectrometer using a multi-turn time-of-flight mass spectrometer, MULTUM-IMG. A mesh pattern image was obtained with spatial resolution of 3 μm. Separation of stigmatic ion images according to the mass-to-charge ratio was also successfully demonstrated with the micro-dot pattern made with crystal violet and methylene blue. An ion image of a micro-dot pattern sample was observed after ten cycles in the multi-turn circuit of MULTUM-IMG, and the pattern of the sample was maintained. Then, stigmatic ion images of the ions of crystal violet and methylene blue produced from the stained section of a brain were observed. An ion image of whole part of a hippocampus (3.25 mm × 1.5 mm) in the brain section can be obtained within 13 minutes.