Congenital disorders of glycosylation (CDG) constitute a group of rare inherited metabolic disorders resulting from mutations in genes involved in the biosynthesis of glycan chains that are covalently attached to proteins or lipids. To date, nearly 200 genes have been identified as responsible for these disorders, with approximately half implicated in N-glycosylation defects. Diagnosis of CDG is primarily achieved through genetic analysis and the identification of glycan abnormalities, referred to as molecular phenotypes. With the increasing use of whole exome and genome sequencing in the investigation of diseases with unknown etiology, the number of cases suspected of CDG is increasing, highlighting the necessity for glycan analysis. Molecular phenotyping in CDG typically targets glycoproteins, with transferrin and apolipoprotein CIII being key representatives of N- and mucin-type O-glycosylation, respectively. Mass spectrometry (MS) provides rapid analysis and yields moderately detailed information, establishing it as a first-line molecular diagnostic tool that complements genetic analysis. Structural anomalies detected by MS can be classified into distinct patterns, which may indicate specific defects within the glycosylation pathway. In cases of CDG types that lack clear molecular phenotypes, characteristic metabolites can often be identified and quantified by MS, further aiding in the diagnostic process. Molecular diagnosis of CDG using MS can be performed with a standard mass spectrometer and a dried blood spot on filter paper, enabling its application in population-based mass screening.

Artificial intelligence (AI) has provided viable methods for retrieving, organizing, and analyzing mass spectrometry (MS) data in various applications. However, several challenges remain as this technique is still in its early, preliminary stages. Critical limitations include the need for more effective methods for identification, quantification, and interpretation to ensure rapid and accurate results. Recently, high-throughput MS data have been leveraged to advance machine learning (ML) techniques, particularly in matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and MS imaging (MSI). The accuracy of AI models is intricately linked to the sampling techniques used in MALDI and MALDI imaging measurements. With the help of artificial neural networks, traditional barriers are being overcome, accelerating data acquisition for different applications. AI-driven analysis of chemical specificity and spatial mapping in two-dimensional datasets has gained significant attention, highlighting its potential impact. This review focuses on recent AI applications, particularly supervised ML in MALDI-TOF MS and MALDI-MSI data analysis. Additionally, this review provides an overview of sample preparation methods and sampling techniques essential for ensuring high-quality data in deep learning-based models.

Amphibians, as one of the leaders of immune resistance, have lived on Earth for hundreds of millions of years. Their dorsal glands produce a cocktail of biologically active peptides that successfully fight microorganisms and even predators. Since this mechanism prevents the development of pathogen resistance, antimicrobial peptides are very promising pharmaceuticals for future generations. Mass spectrometry is the most powerful tool for sequencing peptides/proteins. For over 30 years of studies in this field, mass spectrometry has resolved all the problems associated with the de novo sequencing of amphibian peptides. This review covers the modern de novo sequencing algorithms that enable achieving complete sequence coverage of all frog peptides, including long ones (up to 46 amino acids). Accurate mass measurements have reliably solved the problem of isobaric amino acids. Moreover, there is no longer any need to carry out any preliminary derivatization procedures such as breaking disulfide bonds or N-terminal acetylation. EThcD and ExD tools with manual spectra interpretation provide an efficient approach for reliable differentiation between isomeric leucine and isoleucine residues in the chain, using secondary w- and d-ions, and they resolve the problems of sequencing inside the intact S–S cycles.

Polyethylene terephthalate (PET) is widely used across various industries owing to its versatility and favorable properties, including application in beverage bottles, food containers, textile fibers, engineering resins, films, and sheets. However, polymer materials are susceptible to degradation from factors such as light, oxygen, and heat. Therefore, it is crucial to understand the structural changes that occur during degradation and the extent of these changes. This report investigates the structural alterations in PET films resulting from ultraviolet (UV) irradiation utilizing pyrolysis–gas chromatography time-of-flight mass spectrometry (Py-GC-TOFMS) and matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOFMS). Using the reactive Py-GC-TOFMS, we estimated the composition of the pyrolysis products resulting from UV degradation through electron ionization, soft ionization, and exact mass measurements. Additionally, artificial intelligence (AI)-based structure analysis was performed to evaluate these compounds’ structures. Notably, most degradation products were not found in the National Institute of Standards and Technology database, underscoring the effectiveness of our approach. Using MALDI-TOFMS analysis, we determine the changes in the end groups before and after UV irradiation. This analysis confirmed the generation of a series of carboxylic acid end groups as a result of degradation, a polymer series not detected by reactive pyrolysis GC-MS. We also explored degradation in the depth direction, demonstrating that degradation progresses gradually to depths of several micrometers. Our findings highlight the importance of employing mass spectrometry techniques for a comprehensive analysis of polymer degradation.

The appearance of the characteristic peak of the hydride-eliminated molecule [M−H]⁺ under a positive ion mode (positive) fast atom bombardment (FAB) ionization condition and liquid-assisted secondary ion mass spectrometry (LSIMS) conditions is known for some compounds and the mechanism of its formation has been investigated. In this study, we investigated the formation mechanism of the hydride-eliminated molecule [M−H]⁺ from 4-substituted-1-(methoxymethyl)benzene under a positive FAB ionization condition. The mass spectra of 4-methoxy-1-(methoxymethyl)benzene (1), 4-methoxy-1-(methoxymethyl-d₂-)benzene (1-d₂), and 4-methoxy-1-(methoxymethyl-d₃)benzene (1-d₃) were measured under the positive FAB conditions. [M−H]⁺ was observed for 1 and 1-d₃, and [M−D]⁺ for 1-d₂, indicating that the site of hydride elimination was the methylene of the 1-(methoxymethyl) moiety. Since [M−H]⁺ was hardly observed under the conditions of positive electron ionization and positive chemical ionization in the gas phase, the hydride elimination is a reaction specific to positive FAB ionization. To examine the contribution of the 4-substituent to the hydride elimination reaction, the mass spectra of (methoxymethyl)benzene (2) and 4-nitro-1-(methoxymethyl)benzene (3) were measured using the positive FAB. The ordering of the relative peak intensity of [M−H]⁺ for [M+H]⁺ in the FAB mass spectra was 1 > 2 > 3, and the results suggest that the electron-donating power of the substituents is an important factor in the formation of [M−H]⁺.

In matrix-assisted laser desorption ionization mass spectrometry, a suitable matrix is often selected for the analyte. Herein, we first developed a novel matrix, alkylated hydroxychalcone (AHC), which has properties similar to alkylated trihydroxyacetophenone (ATHAP) (Anal. Chem., 85: 9444–9448, 2013) developed as a matrix for hydrophobic peptides. However, the sample-to-sample reproducibility was low because of the poor crystallinity of AHC. The crystalline morphology of AHC changed when AHC/2,5-dihydroxybenzoic acid (DHB) was used as a binary matrix. As a result, the use of AHC/DHB improved sample-to-sample reproducibility and increased sensitivity for hydrophobic peptides. Mass imaging indicated that these results were due to an increased number of sweet spots wherein the analytes were detected as ion peaks, in a matrix/analyte crystal spot.

Light stabilizers are additives that are widely used to improve the lifespan and performance of polymer materials. To develop advanced polymer materials, analytical techniques investigate the degradation mechanisms and distribution of additives in polymers are crucial. Herein, two extraction–ionization methods were used: tapping-mode scanning probe electrospray ionization (t-SPESI) and liquid extraction surface analysis (LESA). The distribution and molecular structure of the photodegradation products were investigated using polyethylene films containing two types of oligomeric hindered amine light stabilizers (o-HALS). In addition, to study the relationship between light irradiation time and the relative amount of photodegradation products, we developed a method for preparing films with multiple photodegradation regions. Mass spectrometry imaging (MSI) using t-SPESI (t-SPESI-MSI) revealed that the signal intensities of HALS decreased with the time of light irradiation, and its degradation products progressively changed. Moreover, tandem mass spectrometry (MS/MS) using LESA (LESA-MS/MS) revealed that degradation products were generated by HALS fragmentation in the polymer film. By integrating these results, we propose multiple and stepwise reactions for the formation of the photodegradation products. Results indicate that the combined use of t-SPESI-MSI and LESA-MS/MS can directly analyze and understand the photodegradation mechanism of o-HALS in polymer materials.

The ion coalescence phenomenon complicates the evaluation of the effective resolution of Fourier-transform mass spectrometers. We propose an approach for confirming the resolution of an electron ionization Fourier-transform mass spectrometer using pairs of organic substances identified by automatically generated formula differences. The proposed method is compared with the search for organic substances in the National Institute of Standards and Technology (NIST) database. Under the given conditions of the mass spectrometer resolution range up to 45000–50000 at 100 m/z, 166 pairs of suitable compounds were found using the proposed method, while a search in the NIST database yielded only 88 pairs of compounds. This enabled the selection of six pairs of organic compounds that were most suitable for confirming the resolution of the high-resolution mass spectrometer using molecular ion peaks, and four pairs of compounds that allowed the resolution to be confirmed using fragment ion peaks. The resolution of the Fourier-transform mass spectrometer designed for gas analysis was experimentally evaluated by analyzing the spectra of a mixture of organic compounds selected using the proposed method.

Isotope dilution mass spectrometry is a widely used method for measuring intracellular metabolite concentrations, relying on the ratio of peak areas between the target compound and its stable isotope–labeled internal standard. For metabolome analysis of microorganisms, comprehensive concentration measurements have been achieved through the preparation of stable isotope–labeled internal standard extracts (SILIS). Methods have been developed to prepare SILIS by extracting crude metabolites from fully ¹³C-labeled bacteria Escherichia coli and yeasts Saccharomyces cerevisiae and Pichia pastoris (Komagataella phaffii). For cost-effective preparation of SILIS, ideal characteristics of host yeasts include rapid cell growth, high biomass production, and significant metabolite accumulation. In this study, suitable yeast species for SILIS production were investigated from diverse candidates. Batch cultures of 15 yeast species from 12 genera were performed in synthetic defined medium, with cells harvested at different growth phases and metabolites extracted using the methanol/chloroform/water method. Metabolomic analysis by liquid chromatography–tandem mass spectrometry revealed the relative concentrations of 65 metabolites. The results demonstrated that S. cerevisiae and Kluyveromyces marxianus in the stationary phase were the most effective for SILIS production of central metabolic intermediates. SILIS production using S. cerevisiae and K. marxianus can be widely applied in standard laboratories because these species are safe, the media are commercially available, and the extraction methods are easily implementable.

Temperature effects on differentiating d-amino acids using the molecular recognition ability of l-tryptophan were investigated by ultraviolet photodissociation spectroscopy in the gas phase. Temperature-dependent ultraviolet photodissociation spectra of hydrogen-bonded protonated clusters of l-tryptophan with arginine, lysine, asparagine, and glutamine enantiomers, generated via electrospray ionization, were obtained using a tandem mass spectrometer containing a variable-temperature ion trap. The spectra at 8 K differed between the amino acids and their enantiomers, indicating that l-tryptophan recognized amino acids and their enantiomers through its hydrogen bonding and electronic structure. The spectral differences observed at 100 K were significantly smaller than those at 8 K. Hot bands and entropic effects at liquid nitrogen cooling temperature prevented the differentiation of d-amino acids. To avoid these contributions in the spectra, cooling of the hydrogen-bonded clusters using a cryogenic refrigerator was necessary to distinguish amino acids and their enantiomers based on the molecular recognition of l-tryptophan.

We report a robotic sheath-flow probe electrospray ionization mass spectrometry system with a new probe design and automated positioning capabilities for pinpoint ambient mass spectrometry. The system integrates a 3-axis Cartesian robot with two cameras: a fixed global camera for coarse positioning and a movable local camera for fine alignment, enabling users to designate sampling spots by mouse-clicking on live images displayed on the graphical user interface. A microcontroller is used for voltage control, current monitoring, and the detection of probe–sample contact. Sampling, transfer, ionization, and cleaning are fully automated under computer control, and the sheath liquid is supplied by a programmable syringe pump to maintain a stable flow rate. The system successfully analyzed aqueous standards, beverages, and water-rich soft materials such as jelly and fruit slices, yielding stable ion signals with negligible carry-over between measurements.

Scorpion venoms contain a variety of peptides that exhibit toxicity toward insects or mammals by acting on ion channels. We previously isolated four insecticidal peptides (Bl-1, Bl-2, Bl-3, and Bl-4) from the venom of Buthacus leptochelys. Among these, the complete amino acid sequence of Bl-1 was determined, whereas only N-terminal partial sequences were obtained for the others. In the present study, we determined the complete sequence of Bl-3 through de novo sequencing of enzymatically digested fragments. The discrimination between Leu and Ile was achieved based on side-chain fragmentation observed under high-energy collision-induced dissociation conditions. Bl-3 was identified as a 65-residue peptide containing four disulfide bonds. During the sequencing analysis, deamidation of the Asn residue at position 30 was observed, which is likely to have occurred after the purification step. Sequence comparison revealed that Bl-3 shares high similarity with α-toxins that act on sodium channels and exhibit nonselective toxicity toward both insects and mammals. These findings suggest that Bl-3 is likely to exert nonselective toxicity through a mechanism similar to that of α-toxins.

Gold nanorods (AuNRs) possess anisotropic optical and electronic properties, primarily determined by their aspect ratio and surface ligands, which make them attractive for applications in sensing, catalysis, and nanomedicine. While these nanorods are typically stabilized using cetyltrimethylammonium bromide (CTAB) to ensure colloidal dispersion, the cytotoxicity and strong surface affinity of CTAB hinder further surface modification through ligand exchange. In this study, we employed matrix-free laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF-MS) to directly monitor the ligand exchange process on AuNRs. This technique enables the detection of intact CTAB, transient intermediates, and final thiol-bound ligands without requiring chemical derivatization. By correlating mass spectral data with ultraviolet–visible–near-infrared absorption and zeta potential measurements, we elucidate a stepwise ligand exchange mechanism in which CTAB is gradually displaced by a thiol-functionalized phosphorylcholine ligand, facilitated by electrostatic interaction with poly(styrene sulfonate). These findings highlight the utility of matrix-free LDI-TOF-MS as a powerful analytical tool for gaining mechanistic insights into ligand exchange reactions at the nanoscale, particularly in aqueous environments.

Pesticide residues in water contamination represent a significant public and political issue due to their harmful effects on the environment, biodiversity, and human health, even at low concentrations. Pesticides are chemically heterogeneous, covering a wide range of LogK_o/w values. Therefore, developing sensitive methods to detect a broad spectrum of hazardous chemicals in aqueous matrices is challenging. Gas and liquid chromatography/high-performance liquid chromatography-mass spectrometry (GC/HPLC-MS) are established tools but typically require pre-concentration steps. Stir bar sorptive extraction (SBSE) is a green, simple, automatable, and HPLC-compatible technique. This study presents a multi-residue method for determining 131 pesticides in mineral water using SBSE followed by HPLC-tandem MS. The selected pesticides, from various chemical classes, were evaluated in fortified ultra-pure and mineral water samples. The method demonstrated excellent sensitivity, with lower limits of quantification ranging from 20 to 50 ng/L for all analytes, enabling detection at trace levels. Selectivity was high (SEL% <20%), and reproducibility was confirmed with RSD% values below 20%. Intra- and interday precision tests revealed RSD% values from 0.23% to 19.81%. Trueness was validated with BIAS% below 20% at all concentrations. Uncertainty values were acceptable, with U% ranging from 1.44% to 49.24%. This SBSE-HPLC-tandem MS method is a robust, efficient, and reliable alternative to traditional approaches for routine monitoring of pesticide residues in drinking water, with quantification limits below regulatory requirements. It offers a suitable tool for public health applications, ensuring reliable pesticide detection in complex water matrices.

In this study, we propose an effective summarization method for mass spectrometry imaging (MSI) data and demonstrate its efficacy. The MSI data used in this study were obtained from thoracic tissue sections of mice, including the thymus. The thymus is a multi-lobed organ composed of cortical and medullary areas, playing a crucial role in T-cell differentiation. By applying MSI to the thoracic region, including the thymus, this study aims to comprehensively visualize changes in molecular localization and metabolic patterns across thoracic organs. MSI data are highly information-rich, making effective summarization and organization challenging. Therefore, we explored a method to organize and visualize the data based on either spatial or m/z values. Specifically, we employed Uniform Manifold Approximation and Projection (UMAP) to project m/z data into 3-dimensional space, followed by k-means clustering to divide it into multiple clusters. This approach enables detailed and comprehensive representation of diverse features. The objective of this study is to identify molecular localizations and patterns that conventional methods may overlook. Furthermore, experimental results demonstrated that the pseudo-color images generated using UMAP highlighted specific m/z values that significantly influence image characteristics. When focusing on thoracic data, spatial segmentation resulted in clearer color differentiation; however, molecular localizations corresponding to blood vessels were not observed. This finding confirms that m/z segmentation is more effective than spatial segmentation in discovering new molecular localizations.

Chlorogenic acids, esters of hydroxycinnamic acids with quinic acid, are abundant plant metabolites with over 400 known derivatives. Due to the limited availability of commercial standards, mass spectrometry fragmentation data are essential for structural identification. We acquired fragmentation spectra of six chlorogenic acid homologs in both positive- and negative-ion modes using direct infusion mass spectrometry. In positive-ion mode, sodiated molecules provided additional structural information in addition to that from protonated molecules, although the difference in substitution positions had minimal effects on fragmentation patterns. In negative-ion mode, fragmentation differed significantly depending on the acyl group substitution position on the quinic acid moiety, enabling isomer differentiation. This positional selectivity in negative-ion fragmentation parallels previous observations with anhydrous monosaccharides and oligosaccharides. Comparative analysis with maltotriose and β-glucan trisaccharides demonstrated that negative-ion mode fragmentation yields more diagnostic ring cleavage information for structural characterization. This study also emphasizes that the adoption of unambiguous IUPAC (International Union of Pure and Applied Chemistry)-based nomenclature is fundamental to ensuring the reliability of mass spectra databases.

I investigated the tandem mass spectrometry (MS/MS) fragmentation of ginsenoside glycosides using matrix-assisted laser desorption/ionization MS for ginsenosides Rg1, Rh1, Rb1, and Rb3, focusing on their sodium adduct molecules [M+Na]⁺. The glycosidic linkage at the C-20 position cleaved more readily than those at C-3 and C-6. These glycosides fragmented on their glucosyl acceptor sides, exhibiting C- and Z-type fragmentation, although generally B/Y-type fragment ions are dominant in MS/MS spectra of neutral oligosaccharides. These results suggest that, due to the hydrophobic triterpene skeleton of the aglycone, sodium cations cannot effectively coordinate with the aglycone moiety.