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.

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.