A combination of direct liquid extraction using a small volume of solvent and electrospray ionization allows the rapid measurement of complex chemical components in biological samples and visualization of their distribution in tissue sections. This review describes the development of such techniques and their application to biological research since the first reports in the early 2000s. An overview of electrospray ionization, ion suppression in samples, and the acceleration of specific chemical reactions in charged droplets is also presented. Potential future applications for visualizing multimolecular environments in biological systems are discussed.

The insertion of ion mobility spectrometry (IMS) between LC and MS can improve peptide identification in both proteomics and phosphoproteomics by providing structural information that is complementary to LC and MS, because IMS separates ions on the basis of differences in their shapes and charge states. However, it is necessary to know how phosphate groups affect the peptide collision cross sections (CCS) in order to accurately predict phosphopeptide CCS values and to maximize the usefulness of IMS. In this work, we systematically characterized the CCS values of 4,433 pairs of mono-phosphopeptide and corresponding unphosphorylated peptide ions using trapped ion mobility spectrometry (TIMS). Nearly one-third of the mono-phosphopeptide ions evaluated here showed smaller CCS values than their unphosphorylated counterparts, even though phosphorylation results in a mass increase of 80 Da. Significant changes of CCS upon phosphorylation occurred mainly in structurally extended peptides with large numbers of basic groups, possibly reflecting intramolecular interactions between phosphate and basic groups.

Recently developed methods of ambient ionization allow the collection of mass spectrometric datasets for biological and medical applications at an unprecedented pace. One of the areas that could employ such analysis is neurosurgery. The fast in situ identification of dissected tissues could assist the neurosurgery procedure. In this paper tumor tissues of astrocytoma and glioblastoma are compared. The vast majority of the data representation methods are hard to use, as the number of features is high and the amount of samples is limited. Furthermore, the ratio of features and samples number restricts the use of many machine learning methods. The number of features could be reduced through feature selection algorithms or dimensionality reduction methods. Different algorithms of dimensionality reduction are considered along with the traditional noise thresholding for the mass spectra. From our analysis, the Isomap algorithm appears to be the most effective dimensionality reduction algorithm for negative mode, whereas the positive mode could be processed with a simple noise reduction by a threshold. Also, negative and positive mode correspond to different sample properties: negative mode is responsible for the inner variability and the details of the sample, whereas positive mode describes measurement in general.

The gas-phase adsorption of N₂ on protonated serine (Ser, C₃H₇NO₃), threonine (Thr, C₄H₉NO₃), glycine (Gly, C₂H₅NO₂), and 2-aminoethanol (C₂H₇NO) was investigated using a tandem mass spectrometer equipped with an electrospray ionization source and a cold ion trap. N₂ molecules were adsorbed on the free X–H (X=O and N) groups of protonated molecules. Gas-phase N₂ adsorption-mass spectrometry detected the presence of free X–H groups in the molecular structures, and was applied to the structural elucidation of small molecules. When the 93 structures with an elemental composition of C₃H₇NO₃ were filtered using the gas-phase N₂ adsorption-mass spectrometry results for Ser, the number of possible molecular structures was reduced to 8 via the quantification of the X–H groups. Restricting and minimizing the number of possible candidates were effective steps in the structural elucidation process. Gas-phase N₂ adsorption-mass spectrometry combined with mass spectrometry-based techniques has the potential for being useful for elucidating the molecular structures of a variety of molecules.

The enantioselectivity of tryptophan (Trp) for amino acids, such as alanine (Ala), valine (Val), and serine (Ser), was investigated using ultraviolet (UV) photoexcitation and tandem mass spectrometry. Product ion spectra of cold gas-phase amino acid enantiomers that were hydrogen-bonded to Na⁺(L-Trp) were measured using a variable-wavelength UV laser and a tandem mass spectrometer equipped with an electrospray ionization source and a cold ion trap. Na⁺(L-Trp), formed via amino acid detachment, and the elimination of CO₂ from the clusters were observed in the product ion spectra. For photoexcitation at 265 nm, the relative abundance of Na⁺(L-Trp) compared to that of the precursor ion observed in the product ion spectrum of heterochiral Na⁺(L-Trp)(D-Ala) was larger than that observed in the product ion spectrum of homochiral Na⁺(L-Trp)(L-Ala). A difference between the Val enantiomers in the relative abundance of the precursor and product ions was observed in the case of photoexcitation at 272 nm. The elimination of CO₂ was not observed for L-Ser for the 285 nm photoexcitation, which was the main reaction pathway for D-Ser. Photoexcited Trp chiral recognition was applied to identify and quantify the amino acid enantiomers in solution. Ala, Val, and Ser enantiomers in solution were quantified from their relative abundances in single product ion spectra measured using photoexcitation at 265, 272, and 285 nm, respectively, for hydrogen-bonded Trp within the clusters.

Mass spectrometry imaging (MSI) without labeling has the potential for faster screening in drug development. Matrix-assisted laser desorption/ionization (MALDI) is typically used, but it has a large matrix size and uneven drug distribution. Surface-assisted laser desorption/ionization (SALDI) using nanoparticles (NPs) may overcome these issues. Here, the influence of NPs, solvent ratio, and order of dropping of NPs on SALDI-MSI of protoporphyrin IX (PpIX), a cancer drug, are reported. A solution of PpIX in a 50% aqueous solution of 50% acetonitrile at a concentration of 10 μM was used. The NPs include ZnO, Fe₃O₄, and four types of TiO₂. The NPs were fabricated by dissolving them on an aqueous 90% acetonitrile solution. Mass spectra were obtained with a time-of-flight mass spectrometer using a Nd:YAG laser at a 355-nm wavelength. The signal intensity using TiO₂ at a 0.5 mg/mL concentration in 50% acetonitrile was increased by 1.6-fold compared to that without TiO₂. Changing the solvent to 90% acetonitrile gave a uniform TiO₂ distribution and a 9-fold increase in the signal intensity for PpIX. Among the four types of TiO₂ with different particle sizes and crystal structures, TiO₂ with a smaller particle size and a rutile crystal structure produced the highest signal intensity. Forming a layer on top of the PpIX also resulted in an increased signal intensity. Hence, SALDI using TiO₂ provides effective ionization of the drug. In the future, we plan to investigate a spray method for the ionization of PpIX using TiO₂ for the MSI of various drugs.

CO₃^−• and O₂^−• are known to be strong oxidizing reagents in biological systems. CO₃^−• in particular can cause serious damage to DNA and proteins by H^• abstraction reactions. However, H^• abstraction of CO₃^−• in the gas phase has not yet been reported. In this work we report on gas-phase ion/molecule reactions of CO₃^−• and O₂^−• with various molecules. CO₃^−• was generated by the corona discharge of an O₂ reagent gas using a cylindrical tube ion source. O₂^−• was generated by the application of a 15 kHz high frequency voltage to a sharp needle in ambient air at the threshold voltage for the appearance of an ion signal. In the reactions of CO₃^−•, a decrease in signal intensities of CO₃^−• accompanied by the simultaneous increase of that of HCO₃⁻ was observed when organic compounds with H–C bond energies lower than ∼100 kcal mol⁻¹ such as n-hexane, cyclohexane, methanol, ethanol, 1-propanol, 2-propanol, and toluene were introduced into the ion source. This clearly indicates the occurrence of H^• abstraction. O₂^−• abstracts H⁺ from acid molecules such as formic, acetic, trifluoroacetic, nitric and amino acids. Gas-phase CO₃^−• may play a role as a strong oxidizing reagent as it does in the condensed phase. The major discharge product CO₃^−• in addition to O₂^−•, O₃, and NO_x^• that are formed in ambient air may cause damage to biological systems.

A time-of-flight mass spectrometer that uses a closed-orbit flight path can achieve a high mass resolving power and a high mass accuracy with a small instrument footprint. It has long been known that a drawback to a closed flight path is an obtained spectrum may contain peaks by ions at a different number of laps. A lower m/z ion may overtake higher m/z ions, resulting in the peak being superimposed on an acquired mass spectrum; therefore, such a mass bandwidth of the analyzer is limited to a narrow range given the current situation. However, recent research has documented a solution to the problem based on careful study of the equation of motion of an ion in a closed-path analyzer. All of the ions in the analyzer remain in motion in orbit by the nature of the closed flight path, thus resulting in a superimposed spectrum with the width of the orbital period of the highest mass in the sample matrix, which contains several different lap numbers. When target ions for the sample are known in advance, the time-of-flight for a given m/z can be determined regardless of the lap number under given analyzer conditions, and peak assignment can be self-validated by comparison to a mass spectrum acquired at a different lap condition. Furthermore, the m/z value for an unknown ion can also be determined by comparing time-of-flight values on spectra acquired at different lap conditions.