Among the most typical posttranslational modifications is glycosylation, which often involves the covalent binding of an oligosaccharide (glycan) to either an asparagine (N-linked) or a serine/threonine (O-linked) residue. Studies imply that the N-glycan portion of a glycoprotein could serve as a particular disease biomarker rather than the protein itself because N-linked glycans have been widely recognized to evolve with the advancement of tumors and other diseases. N-glycans found on protein asparagine sites have been especially significant. Since N-glycans play clearly defined functions in the folding of proteins, cellular transport, and transmission of signals, modifications to them have been linked to several illnesses. However, because these N-glycans’ production is not template driven, they have a substantial morphological range, rendering it difficult to distinguish the species that are most relevant to biology and medicine using standard techniques. Mass spectrometry (MS) techniques have emerged as effective analytical tools for investigating the role of glycosylation in health and illness. This is due to developments in MS equipment, data collection, and sample handling techniques. By recording the spatial dimension of a glycan’s distribution in situ, mass spectrometry imaging (MSI) builds atop existing methods while offering added knowledge concerning the structure and functionality of biomolecules. In this review article, we address the current development of glycan MSI, starting with the most used tissue imaging techniques and ionization sources before proceeding on to a discussion on applications and concluding with implications for clinical research.

A novel ionization technique named medium vacuum chemical ionization (MVCI) mass spectrometry (MS), which is a chemical ionization using oxonium (H₃O⁺) and hydroxide (OH⁻) formed from water, has excellent compatibility with the supercritical fluid extraction (SFE)/supercritical fluid chromatography (SFC). We have studied a method to determine free fatty acids (FFAs) in a small section of bovine liver tissue using SFE/SFC–MVCI MS analysis without further sample preparation. A series of FFA molecules interact with the C18 stationary phase, exhibiting broad chromatographic peaks when using a non-modified CO₂ as the mobile phase. It can be optimized by adding a small content of methanol to the mobile phase as a modifier; however, it may dampen the ionization efficiency of MVCI since the proton affinity of methanol is slightly higher than water. We have carefully evaluated the modifier content on the ion detection and column efficiencies. The obtained result showed that an optimized performance was in the range of 1 to 2% methanol-modified CO₂ mobile phase for both column efficiency and peak intensity. Higher methanol content than 2% degrades both peak intensity and column efficiency. Using optimized SFC conditions, a section of bovine liver tissue sliced for 14 µm thickness by 1 mm square, which is roughly estimated as about 3300 hepatocytes, was applied to determine 18 FFAs amounts for carbon chains of C12–C24. An amount of each tested FFA was estimated as in the range of 0.07 to 2.6 fmol per cell.

In metabolomic analysis, one of the most commonly used techniques to support the detection sensitivity and quantitation of mass spectrometry is combining it with liquid chromatography. Recently, we developed a method that enables comprehensive single-run measurement of hydrophilic metabolites using unified-hydrophilic interaction/anion exchange liquid chromatography/high-resolution mass spectrometry (unified-HILIC/AEX/HRMS) with a polymer-based mixed amines column (Gelpack GL-HilicAex). However, the importance of stationary phase functional groups and mobile phase conditions for the separation mechanisms and sensitive detection in unified-HILIC/AEX/HRMS is not yet fully understood. This study aimed to understand the importance of the mobile and stationary phases in unified-HILIC/AEX/HRMS. Two different alkali-resistant polymer-based amines-modified columns (Gelpack GL-HilicAex, primary, secondary, tertiary, and quaternary amine-modified polyglycerol dimethacrylate gel; Asahipak NH2P-50 2D, secondary amine-modified polyvinyl alcohol gel) and two eluents (acetonitrile and ammonium bicarbonate solution, pH 9.8) were used for comparative validation. A comparison of mobile phase conditions using both columns confirmed that the two-step separation from HILIC to AEX characteristic of unified-HILIC/AEX requires a linear gradient condition from acetonitrile to nearly 50% water and AEX with up to 40 mM bicarbonate ions. We found that when alkali-resistant hydrophilic polymer packing materials are modified with amines, unified-HILIC/AEX separation can be reproduced if at least one secondary amine associated with the amine series is present in the stationary phase. Furthermore, the difference in sensitivity in the HILIC and AEX modes owing to the different columns indicates the need for further improvements in the mobile phase composition and stationary phase.

The collision-induced dissociation (CID) behaviors of protonated molecules of anabaenopeptins, a group of cyanobacterial cyclic peptides, were investigated in detail using liquid chromatography-tandem mass spectrometry. Although anabaenopeptin A and B share a macrocyclic peptide structure, they give strikingly different fragmentation patterns; the former gives a variety of product ions including cleavages in the cyclic peptide structure, which is useful for structural analysis; whereas the latter gives far fewer product ions and no fragmentation in the cyclic moiety. Energy-resolved CID experiments clarified the mechanism behind the striking difference attributable to the difference in exocyclic amino acid residues, Tyr or Arg. The guanidino group in Arg-containing analogue, anabaenopeptin B, should be by far the most preferred protonation site; the proton would be sequestered at the guanidino group in the protonated molecule, with the lack of proton mobility prohibiting opening of the charge-directed fragmentation channels in the cyclic moiety. Enzymatic hydrolysis of the guanidino group to give citrullinated-anabaenopeptin B restored proton mobility. The fragmentation pattern of the citrullinated peptide became almost identical to that of anabaenopeptin A. The observed fragmentation behaviors of these cyclic peptides were consistent with those of linear peptides, which have been well understood based on the mobile proton model.

Aims: The purpose of this study is to establish a novel diagnosis system in early acute coronary syndrome (ACS) using probe electrospray ionization-mass spectrometry (PESI-MS) and machine learning (ML) and to validate the diagnostic accuracy.

Methods: A total of 32 serum samples derived from 16 ACS patients and 16 control patients were analyzed by PESI-MS. The acquired mass spectrum dataset was subsequently analyzed by partial least squares (PLS) regression to find the relationship between the two groups. A support vector machine, an ML method, was applied to the dataset to construct the diagnostic algorithm.

Results: Control and ACS groups were separated into the two clusters in the PLS plot, indicating ACS patients differed from the control in the profile of serum composition obtained by PESI-MS. The sensitivity, specificity, and accuracy of our diagnostic system were all 93.8%, and the area under the receiver operating characteristic curve showed 0.965 (95% CI: 0.84–1).

Conclusion: The PESI-MS and ML-based diagnosis system are likely an optimal solution to assist physicians in ACS diagnosis with its remarkably predictive accuracy.

Electrospray ionization mass spectrometry of neat undiluted ionic liquid (IL) and the analysis of protein with the doping of IL were performed using high-pressure electrospray. The use of disposable micropipette tips as emitters eased the handling of viscous and easy-to-clog samples and improved the reproducibility of the measurement. A high-pressure operation enabled the stable electrospray of the highly conductive IL from these relatively large bore emitters. The measurement of the current–voltage relationship of 1-ethyl-3-methylimidazolium tetrafluoroborate (Emim BF₄) revealed an unusual negative differential resistance that has not been seen in the typical atmospheric or high-pressure electrospray. Mass spectrometric analysis of this IL also showed the characteristic response of various ion species with the emitter voltage. When added to the commonly used protein solution, the mass spectrum also showed protein peaks that correspond to the adduction of fluoroboric acid molecules (HBF₄).

Mass spectrometry imaging (MSI) is a technique that visualizes the distribution of molecules by ionizing the components on the surface of a sample and directly detecting them. Previously, MSI using hair has primarily been used in the forensic field to detect illegal drugs. On the other hand, there are few examples of using this technology for health monitoring. In this study, hair and clinical data were collected from 24 subjects, and the correlation between blood cholesterol levels and cholesterol detected from cross-sectional hair slices was analyzed. As a result, a positive correlation with a correlation coefficient of 0.43 was observed between blood cholesterol and cholesterol detected from hair. Furthermore, when comparing the results of fluorescence staining (FS) of hair cholesterol with Filipin III and the MSI results, it was found that while FS could visualize detailed hair structures, there were cases where the results differed from MSI, possibly due to some cholesterol loss during the staining process. In the future, if various disease biomarkers can be detected using hair MSI, it could potentially become a non-invasive diagnostic method.

Skin dryness and irritant contact dermatitis induced by the prolonged use of surgical gloves are issues faced by physicians. To address these concerns, manufacturers have introduced surgical gloves that incorporate a moisturizing component on their inner surface, resulting in documented results showing a reduction in hand dermatitis. However, the spatial distribution of moisturizers applied to surgical gloves within the integument remains unclear. Using matrix-assisted laser desorption/ionization (MALDI)–mass spectrometry imaging (MSI), we investigated the spatial distribution of moisturizers in surgical gloves within artificial membranes. Recently, dermal permeation assessments using three-dimensional models, silicone membranes, and Strat-M have gained attention as alternative approaches to animal testing. Therefore, in this study, we established an in vitro dermal permeation assessment of commercially available moisturizers in surgical gloves using artificial membranes. In this study, we offer a methodology to visualize the infiltration of moisturizers applied to surgical gloves into an artificial membrane using MALDI–MSI, while evaluating commercially available moisturizer-coated surgical gloves. Using our penetration evaluation method, we confirmed the infiltration of the moisturizers into the polyethersulfone 2 and polyolefin layers, which correspond to the epidermis and dermis of the skin, after the use of surgical gloves. The MSI-based method presented herein demonstrated the efficacy of evaluating the permeation of samples containing active ingredients.

The areoles and spines of cacti can be used to desorb ions of ionic liquids (ILs) by the mere action of an electric field into the atmospheric pressure (AP) interface of a mass spectrometer. The small cactus species Opuntia microdasys bears numerous very fine hairs on its areoles and tiny sharp spines that appeared suited to serve as needle electrodes sharp enough for field desorption of ions to occur. In fact, positive and negative ions of four ILs could be desorbed by a process analogous to AP field desorption (APFD). In contrast to APFD where activated field emitters are employed, the ILs were deposited onto one or two adjacent areoles by applying 1–3 µL of a dilute solution in methanol. After evaporation of the solvent, the cactus was positioned next to the spray shield electrode of a trapped ion mobility-quadrupole-time-of-flight instrument. Desorption of IL cations and IL anions, respectively, did occur as soon as the electrode was set to potentials in the order of ±4.5 kV, while the cactus at ground potential was manually positioned in front of the entrance electrode to bring the areole covered with a film of the sample into the right position. Neither did mixing of ILs occur between neighboring areoles nor did the cactus suffer any damage upon its use as a botanical field emitter.