Atmospheric pressure electrospray had been used in previous studies to generate massive water droplet ion beams, and the beams successfully achieved efficient desorption/ionization of biomolecules, low damage etching of polymers and nonselective etching of metal oxides. However, this droplet ion beam was not practical as a primary ion beam for surface analysis instruments because it required differential pumping and lacked adequate beam current and density. To improve the beam performance, we have proposed to use vacuum electrospray of aqueous solutions as a beam source, and developed a technique for producing a stable electrospray of aqueous solution in vacuum. We also designed a prototype of a vacuum electrospray droplet ion gun, and measured the beam properties. Finally, the applicability of this ion gun in secondary ion mass spectrometry is discussed.

Ion mobility experiments coupled with electrospray ionization (ESI) were conducted to evaluate the folding states of bovine carbonic anhydrase 2 (CA2) under three different pH conditions. Collision cross-section (CCS) of the CA2 ions generated by ESI revealed the presence of six discrete conformers in the gas phase under the conditions employed in this study. The CCS of the most extended conformer was three times larger than that of the most compact one. The charge state distribution of the CA2 ions was indicative of three conformers being present. Although there was consistency in conformer assignment conducted by CCS and charge state distribution, the CCS measurement was shown to be more effective because the information obtained provided more detailed knowledge of the conformation of the protein.

We report on the development of a software program named iQuant2 which creates visual images from two-dimensional signal intensity data obtained by a laser ablation-ICP-mass spectrometry (LA-ICPMS) technique. Time-resolved signal intensity profiles can be converted to position resolved signal intensity data based on the rastering rate (μm s⁻¹) of the laser ablation. Background signal intensities obtained without laser ablation (gas blank) are used as the background, and all of the blank-subtracted intensity data can be used for the imaging analysis. With this software, deformation of the created image can be corrected visually on a PC screen. The line profile analysis between the user-selected points can be observed using the iQuant2 software. To accomplish this, data points on the profile line were automatically calculated based on the interpolation between the analysis points. The resulting imaging data can be exported and stored as JPEG, BMP or PNG formats for further processing. Moreover, a semi-quantitative analysis can be made based on the coupling of the external correction of the RSF (relative sensitivity factor) using NIST SRM610 with normalization of the corrected signal intensity data being 100%. The calculated abundance data for major elements are in reasonable agreement with the values obtained by electron probe micro analyzer (EPMA). With the software developed in this study, both the rapid imaging and semi-quantitative determinations can be made.

Current bottleneck of comprehensive non-target metabolite identification is insufficient spectral library. Many research groups have tried to build a theoretical product ion spectral library independent of measurement condition or settings, but mechanisms of metabolite fragmentation are not fully clarified. To achieve the mechanistic prediction of metabolite fragmentation which covers a wide range of metabolites, we will discuss utilization of physicochemical calculation. We introduce bonding patterns, which include two bound atoms and chemical groups adjacent to the bond. Cleavage of each bonding pattern is simulated and its activation energy is precisely calculated with quantum chemistry and assigned on metabolites. By tracing low-energy bond cleavages, fragmentation of a dipeptide molecule is successfully predicted. Prediction on another metabolite requires some additional features to fully reproduce its experimentally observed product ions. Physicochemical calculation shows its promising ability to predict fragmentation pathways only from metabolite structures, while required improvements suggested by comparison between our prediction and standard spectra stored in database are also discussed. Moreover, to construct a prediction strategy which withstands the vast metabolite space, we have to build a comprehensive list of bonding patterns and their activation energy. As theoretically possible bonding patterns are huge in number, proper simplification of the patterns must be implemented. We will discuss how to achieve it in addition to the prediction results.

Cancer cells often respond to chemotherapeutic inhibitors by redirecting carbon flow in the central metabolism. To understand the metabolic redirections of inhibitor treatment on cancer cells, this study established a ¹³C-metabolic flux analysis (¹³C-MFA)-based method to evaluate metabolic redirection in MCF-7 breast cancer cells using mass spectrometry. A metabolic stationary state necessary for accurate ¹³C-MFA was confirmed during an 8–24 h window using low-dose treatments of various metabolic inhibitors. Further ¹³C-labeling experiments using [1-¹³C]glucose and [U-¹³C]glutamine, combined with gas chromatography-mass spectrometry (GC-MS) analysis of mass isotopomer distributions (MIDs), confirmed that an isotopic stationary state of intracellular metabolites was reached 24 h after treatment with paclitaxel (Taxol), an inhibitor of mitosis used for cancer treatment. Based on these metabolic and isotopic stationary states, metabolic flux distribution in the central metabolism of paclitaxel-treated MCF-7 cells was determined by ¹³C-MFA. Finally, estimations of the 95% confidence intervals showed that tricarboxylic acid cycle metabolic flux increased after paclitaxel treatment. Conversely, anaerobic glycolysis metabolic flux decreased, revealing metabolic redirections by paclitaxel inhibition. The gap between total regeneration and consumption of ATP in paclitaxel-treated cells was also found to be 1.2 times greater than controls, suggesting ATP demand was increased by paclitaxel treatment, likely due to increased microtubule polymerization. These data confirm that ¹³C-MFA can be used to investigate inhibitor-induced metabolic redirection in cancer cells. This will contribute to future pharmaceutical developments and understanding variable patient response to treatment.

2,6-Dichloro-1,4-benzoquinone (DCBQ), a highly toxic and carcinogenic disinfection by-product, was degraded during the electrospray process by elevating the source temperature. This unexpected finding inspired us to use heating to degrade DCBQs in drinking water. The results show that about 99% of DCBQs in the drinking water were degraded in one minute by heating to 100°C with room light irradiation. Therefore, a conclusion can be drawn that heating enables the degradation of DCBQs in drinking water.

Oligonucleotide-based therapeutics such as antisense oligonucleotides, small interfering RNAs (siRNAs), decoy and aptamer have been extensively developed. To investigate the pharmacokinetics of oligonucleotide therapeutics, it is common to label a radioisotope in a nucleic acid and visualize it. However, if the labeled terminal nucleotide is decomposed by a nuclease in vivo, only the labeled nucleotide is detected, and it is impossible to observe the nucleic acid exhibiting the drug effect. The distribution of biomolecules, such as phospholipids, proteins, and glycolipids, can be obtained and visualized without labeling using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS). MALDI-IMS is also used in pharmacokinetic analysis to visualize a parent drug and its metabolites simultaneously. In this study, we reported a methodology for oligonucleotides analysis by MALDI-IMS. When phosphorothioate antisense oligonucleotide was administered into the eyeball of rats, it reached the retina after 30 min without undergoing decomposition by nucleases.

A sheet-like ultraviolet (UV) probe laser is used to investigate the ejection and propagation of ion packets of matrix CHCA, which are produced by matrix-assisted laser desorption and ionization (MALDI). Laser irradiation of the expanding MALDI plume induced photodissociation of the CHCA-related ions, which existed in a sheet-like volume, leading to their absence in their MALDI signal profiles. The MALDI spectra were measured under varying conditions: the temporal delay of the lasers and the distance of the sheet-like probe laser from the MALDI sample surface. It was found that the center of the (CHCA)H⁺ packets were ejected at 46±11 ns after MALDI laser irradiation, while the (CHCA)₂H⁺ packets were ejected at 64±12 ns, regardless of the magnitude of acceleration static high-voltage in 3.5–5.5 kV. This suggests that (CHCA)₂H⁺ is formed by a proton transfer reaction from (CHCA)H⁺ to (CHCA)₂ in the heated condensed phase and/or near the surface. This study represents the first experimental determination of ion ejection time in the MALDI process, which is also applicable to other species in the MALDI plume.