Desorption ionization using through-hole alumina membrane offers higher reproducibility than 2,5-dihydroxybenzoic acid, a widely used matrix in Fourier transform ion cyclotron resonance mass spectrometry imaging analysis.

RATIONALE: DIUTHAME (desorption ionization using through-hole alumina membrane), a recently developed matrix-free ionization-assisting substrate, was examined for reproducibility in terms of mass accuracy and intensity using standard lipid and mouse brain sections. The impregnation property of DIUTHAME significantly improved the reproducibility of mass accuracy and intensity compared with 2,5-dihydroxybenzoic acid (DHB). METHODS: Frozen tissue sections were mounted on indium tin oxide-coated glass slides. DIUTHAME and DHB were applied to individual sections. Subsequently, a solution of a phosphatidylcholine standard, PC(18:2/18:2), was poured onto the DIUTHAME and matrix. Finally, the samples were subjected to laser desorption ionization coupled with Fourier transform ion cyclotron resonance mass spectrometry. The reproducibility was tested by calculating the mean ± standard deviation values of mass errors and intensities of individual ion species. RESULTS: Analysis of the PC(18:2/18:2) standard showed significantly (p < 0.01) lower mass error for DIUTHAME-MS than for MALDI-MS. Endogenous PC(36:4) analysis in mouse brain section also showed significantly (p < 0.05) lower mass errors for DIUTHAME-MS. Furthermore, we investigated the mass error of some abundant lipid ions in brain sections and observed similar results. DIUTHAME-MS displayed lower signal intensity in standard PC analysis. Interestingly, it offered higher signal intensities for all the endogenous lipid ions. Lower fluctuations of both mass accuracies and signal intensities were observed in DIUTHAME-MS. CONCLUSIONS: Our results demonstrated that DIUTHAME-MS offers higher reproducibility for mass accuracies and intensities than MALDI-MS in both standard lipid and mouse brain tissue analyses. It can potentially be used instead of conventional MALDI-MS and mass spectrometry imaging analyses to achieve highly reproducible data for mass accuracy and intensity.

Rapid Fingerprinting of High-Molecular-Weight Polymers by Laser Desorption-Ionization Using Through-Hole Alumina Membrane High-Resolution Mass Spectrometry.

Residual acid found in the desorption ionization using through-holes alumina membranes (DIUTHAME) induces a reproducible protonation/in-source dissociation of polymers made of ester, amide, or siloxane moieties during their surface-assisted laser desorption ionization (SALDI) mass analysis. Deposited on the DIUTHAME chips in solution (solvent-based) or in pure form by melting the polymer powder in situ (solvent-free), high-molecular-weight nylons, silicone, or functionalized celluloses among other polymers are instantly fingerprinted by laser DIUTHAME high-resolution mass spectrometry (MS) with specific patterns resembling their direct analysis in real-time (DART) single-stage or tandem mass spectra. Depending on the polymer, two main types of fingerprints are observed with either the protonated monomer or product ions revealing the nature of the repeating unit or its functionalization. This technique allows a rapid molecular analysis of industrial homopolymers regardless of their molecular weight and complementary to DART with simple or no sample preparation and also promisingly applicable for copolymers.

DIUTHAME enables matrix-free mass spectrometry imaging of frozen tissue sections.

RATIONALE: A recently developed matrix-free laser desorption/ionization method, DIUTHAME (desorption ionization using through-hole alumina membrane), was examined for the feasibility of mass spectrometry imaging (MSI) applied to frozen tissue sections. The permeation behavior of DIUTHAME is potentially useful for MSI as positional information may not be distorted during the extraction of analytes from a sample. METHODS: The through-hole porous alumina membranes used in the DIUTHAME chips were fabricated by wet anodization, were 5 µm thick, and had the desired values of 200 nm through-hole diameter and 50% open aperture ratio. Mouse brain frozen tissue sections on indium tin oxide (ITO)-coated slides were covered using the DIUTHAME chips and were subjected to MSI experiments in commercial time-of-flight mass spectrometers equipped with solid-state UV lasers after thawing and drying without matrix application. RESULT: Mass spectra and mass images were successfully obtained from the frozen tissue sections using DIUTHAME as the ionization method. The mass spectra contained rich peaks in the phospholipid mass range free from the chemical background owing to there being no matrix-derived peaks in that range. DIUTHAME-MSI delivered high-quality mass images that reflected the anatomy of the brain tissue. CONCLUSIONS: Analytes can be extracted from frozen tissue by capillary action of the through-holes in DIUTHAME and moisture contained in the tissue without distorting positional information of the analytes. The sample preparation for frozen tissue sections in DIUTHAME-MSI is simple, requiring no specialized skills or dedicated apparatus for matrix application. DIUTHAME can facilitate MSI at a low mass, as there is no interference from matrix-derived peaks, and should provide high-quality, reproducible mass images more easily than MALDI-MSI.

Molecular characterization of polyethylene oxide based oligomers by surface-assisted laser desorption/ionization mass spectrometry using a through-hole alumina membrane as active substrate.

RATIONALE: Molecular characterization of industrial oligomeric products is performed using surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS), termed desorption ionization using a through-hole alumina membrane (DIUTHAME). This paper describes the unique feature of a DIUTHAME chip applying active SALDI, which generates specific types of fragments of polyglycol samples. METHODS: Polyethylene oxide (PEO) and PEO-based materials were subjected to SALDI-MS. The influence of the presence or absence of a cationization salt on the mass spectrum was investigated. The resulting mass spectra composed of fragment ions were compared with those obtained by collision-induced dissociation (CID)-MS/MS. The specific fragment ions generated using the DIUTHAME chip were further subjected to high-energy CID-MS/MS. RESULTS: The addition of a cationization salt resulted in SALDI mass spectra with fewer fragment peaks. The mass spectra obtained without adding the cationization salt were composed of many more fragment ions caused by in-source decay. The fragmentation pattern was similar to that seen with low-energy CID. The resulting fragment ions were formed by selective cleavage at the C-O bond. High-energy CID-MS/MS can be performed for the specific fragment ions generated by in-source decay fragmentation. CONCLUSIONS: Molecular characterization of PEO-based oligomers by SALDI-MS using the DIUTHAME chip was successfully demonstrated. The selective fragmentation and high-energy CID-MS/MS of the in-source decay fragments made it possible to provide more detailed structural information. This unique feature of DIUTHAME gives it potential for use in new molecular characterization techniques.

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).

Quantitative analysis capability of DIUTHAME mass spectrometry verified by acetylcholinesterase enzyme-catalyzed reaction assays.

Matrix-assisted laser desorption/ionization (MALDI) is advantageous for mass spectrometry applications where throughput is important. However, quantitative analysis is essentially problematic for MALDI-MS whose results depend on the intrinsically stochastic microcrystalline state of the matrix. High-throughput screening (HTS) of drug candidates is a typical example that requires high throughput. The application of MALDI-MS to HTS, which is quantitative analysis, imposes restrictions on designing an experimental system. Surface-assisted laser desorption/ionization (SALDI) methods, which do not depend on matrix crystal formation, are expected to be applied to quantitative analyses such as HTS. A recently developed one type of SALDI methods, desorption ionization using through hole alumina membrane (DIUTHAME), possesses a distinct feature that the surface microstructure effective for ionization is formed by through holes. In this study, the quantitative analysis capability of DIUTHAME was verified by applying DIUTHAME to enzyme-catalyzed reaction measurements, which are also used for HTS. Quantitative DIUTHAME-MS was conducted on various conditions of acetylcholinesterase-catalyzed reaction solutions containing cow milk as a substitute of biological media. Even for the enzyme-catalyzed reaction solutions containing complex additives that make the quantitative analysis extremely difficult, DIUTHAME based on the through hole structure enables quantitative measurements of the analytes by applying the reaction solutions to the back side of the laser exposed surface. In comparison with those obtained by MALDI-MS, the results obtained by DIUTHAME-MS showed less variability of data and delivered a better linearity of the Lineweaver-Burk plots and a more reasonable value of the Michaelis constant. Accordingly, it was demonstrated that DIUTHAME-MS possesses the quantitative analysis capability much better than that of MALDI-MS.

Surface-assisted Laser Desorption/ionization Mass Spectrometry Analysis of the Glycolipid Biosurfactants, Mannosylerythritol Lipids, Using an Ionization-assisting Substrate.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a promising tool for the screening of glycolipid-type biosurfactants (BSs) from a crude extract of microbial products. However, it is unsuitable for the detection of lower molecular weight products because the observed ions are overlapped with matrix-derived ions at lower mass range. In this study, we applied a "matrix-free" surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) analysis using a through-hole alumina membrane as an ionization-assisting substrate. Using this method, we could detect a variety of lower molecular weight products in an extract of a glycolipid BS producer with good sensitivity. In addition, the culture solution could be analyzed directly by this method.

Feasibility of Acetylcholinesterase Reaction Assay Monitoring in DIUTHAME-MS.

DIUTHAME (desorption ionization using through hole alumina membrane) is a novel matrix-free laser desorption/ionization method that enables highly reproducible acquisition of mass spectra. This study aims to evaluate the applicability of DIUTHAME to the acetylcholinesterase reaction assay (AChE assay) commonly used in high-throughput screening (HTS) in the drug discovery process. The commercially available 9-ch DIUTHAME chip was applied to a series of AChE assays prepared with different reaction times. Numerous positive-mode TOF mass spectra were acquired from the raster-scanned sample spots of the AChE assays to analyze the progress of the enzyme reaction and to perform mass spectrometry imaging of the sample spots. The reaction kinetics plots obtained by DIUTHAME were found to reflect the time course of reaction progression as much as those obtained by MALDI and were found to have less error variation than MALDI. DIUTHAME allowed the already irradiated sample spots to be reused to reproduce the reaction kinetics plots by the second measurement conducted a week after, whereas MALDI was not able to properly reproduce the kinetics plots by remeasuring the irradiated sample spots. In DIUTHAME, which does not require a matrix, the experimental procedure for measuring AChE assay becomes extremely simple. DIUTHAME is potentially able to provide more precise AChE reaction kinetics plots than what have been accomplished by MALDI.

Simple Pretreatment for the Analysis of Additives and Polymers by Surface-Assisted Laser Desorption/Ionization Mass Spectrometry Using a Through-Hole Alumina Membrane as a Functional Substrate.

The analysis of additives and polymers was performed by desorption ionization using through-hole alumina membrane (DIUTHAME) as a functional substrate for both sample pretreatment and surface-assisted laser desorption/ionization (SALDI) mass spectrometry. Using the unique absorbing/filtering capabilities of DIUTHAME and investigating the solubility of analytes/bulk materials in some solvents, three pretreatment techniques were demonstrated with (1) the selective removal of hydrophilic poly(ethylene oxide) (PEO)-based components from a "PEO-monostearate" sample, (2) the on-chip filtration of solubilized decabromodiphenylether (DBDE) from a solution of polystyrene that had been preliminarily precipitated, and (3) the on-chip extraction of antioxidants (Irganox 1010, Irgafos 168, and dimyristyl 3,3'-thiodipropionate) from a suspension of polypropylene powder or from the powder itself. The extracted analytes were further mass-analyzed using a spiral high-resolution time-of-flight analyzer to assess their elemental composition or molecular distribution.

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.

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.

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.