The gas-phase environment of a mass spectrometer is the ideal system to benchmark advanced computational methods, as the properties of ionic species derived from mass spectrometry experiments are identical to the target of (quantum) computational predictions. This is particularly applicable for gas-phase ion spectroscopy techniques, where lasers are interfaced with mass spectrometers to gain further information about isolated ions. Complementing experimental results with computational findings allows one to increase the value of the results and to better interpret the data. The calculation of isolated particles in the gas phase is straightforward and requires fewer approximations than more complex systems. This review serves to give a brief overview of where quantum chemistry and mass spectrometry mutually benefit each other. The main topics covered in this review include fragmentation methods, ion–molecule reactions, ion mobility-mass spectrometry, and ion spectroscopy methods.

We have developed a new analytical technique for the detection of volatile organic compounds (VOCs) using inductively coupled plasma tandem mass spectrometry (ICP-MS/MS). Eight VOCs of various polarities (acetic acid, 2-butanone, pyridine, 2-methylfuran, ethylene, benzene, toluene, and limonene) were introduced into the collision/reaction cell (CRC) of the ICP-MS/MS, bypassing the ICP ion source. This approach enabled a softer ionization than the typical ICP, allowing the detection of the molecules in their intact form. In this study, to explore the potential of cationization as a soft ionization approach, the interaction of the above VOCs with various elements (i.e., Li, Be, Na, Mg, V, Co, Ni, Cu, Zn, As, Rb, Sr, Y, Ru, Pd, Ag, Cd, Cs, Ba, Pt, and Hg) was investigated. The production ratio of cation adducts was evaluated using benzene as a model compound by monitoring [C₆H₆ + E]⁺/E⁺ values (E represents each element). The resulting [C₆H₆ + E]⁺/E⁺ showed a wide variation, covering ranges from 0.0001% to 1%, and the elements with ionization energies between 650 and 900 kJ mol⁻¹ exhibited the highest [C₆H₆ + E]⁺/E⁺ values. The data obtained here revealed that several elements, including Co, Ni, Cu, Ru, Pd, and Ag, can be suitable elements for cationization. The reduction in fragmentation resulted in easier detection of specific compounds based on their m/z values. To demonstrate the practicality of the present technique, several VOCs released from coffee beans through laser-induced evaporation were monitored. The data obtained here envisage a possibility that the detection of VOCs through cationization achieved in ICP-MS/MS can become an effective choice as a rapid analytical tool for VOCs in solid samples.

Information on candidate biomarker metabolites identified in recent disease biomarker discovery research is expected to play a key role in the future of personalized and precision medicine. Liquid chromatography mass spectrometry (LC/MS) is a powerful method for metabolomic analysis due to its comprehensive coverage and high detection sensitivity. However, the suitability of LC/MS methods for the identification and quantification of hydrophilic metabolites remains debatable. Here, we evaluated the performance of LC/MS methods combining four types of LC [hydrophilic interaction chromatography (HILIC), ion chromatography (IC) with an anion-exchange (AEX) column (AEX-IC), reversed-phase LC (RPLC) with a pentafluorophenylpropyl (PFPP) column (PFPP-RPLC), and unified-hydrophilic interaction AEX LC (unified-HILIC/AEX)], using the same Orbitrap mass spectrometer, with the aim of integrating future human plasma metabolome data. First, we conducted a qualitative performance evaluation of four LC/MS methods, HILIC/MS, AEX-IC/MS, PFPP-RPLC/MS, and unified-HILIC/AEX/MS, by analyzing 511 hydrophilic metabolite standards and NIST Standard Reference Material (SRM) 1950 (Metabolites in Frozen Human Plasma). The evaluation focused on metabolome coverage, peak width, sensitivity, and separation performance of isomers. Next, we thoroughly evaluated the quantitative performance of the four analytical methods for 63 hydrophilic metabolites in SRM 1950 using a stable isotope-labeled internal standard (SILIS) mixture derived from ¹³C-labeled Escherichia coli extracts. Furthermore, we successfully estimated new concentration values for 29 metabolites without certified values in SRM 1950 using quantitative data from the four LC/MS methods. We objectively evaluated the performance of the four LC/MS methods and demonstrated that absolute quantification using SILIS is effective for integrating hydrophilic metabolite data in metabolomics.

Ambient mass spectrometry (AMS) enables real-time analysis without sample preparation, yet atmospheric pressure corona discharge ionization (APCI)-like ion sources can oxidize analytes in-source, obscuring spectral interpretation. We investigated α-pinene using atmospheric pressure corona discharge ionization mass spectrometry (APCDI-MS; one of APCI-like AMS) and, when needed, coupled gas chromatography (GC) to separate pre-existing oxidation products from species formed inside the ion source. By comparing adduct formation and product-ion patterns, we show that oxygenated ions from primary (pre-source) and secondary (in-source) oxidation display similar ion formation behavior, notably ready NH₄⁺-adduct formation, whereas fragment ions rarely form NH₄⁺ adducts. GC-based characterization of in-source oxidation provides features that assist interpretation of APCI-like AMS spectra acquired without chromatography.

Carnitine (CAR) is an essential compound for animals and plays several physiological roles related to energy production. These functions are possessed by only the l-forms, while the d-forms are known to inhibit the uptake of the l-forms, thus causing a CAR deficiency. Therefore, it is required to determine dl-CAR in various samples such as foods, supplements, and drugs by an enantioselective analytical method. In this study, we developed a chiral liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using derivatization. The CAR enantiomers derivatized with 4-nitrophenylhydrazine were successfully separated on a CHIRALPAK ZWIX (−) column (resolution = 1.64) and detected with high sensitivity and selectivity by MS/MS. The developed method was well validated and applied to the analysis of eight kinds of foods and an l-CAR supplement. Only the l-forms were found in all the tested food samples and were abundant in meat, milk, and yogurt. The system was also applicable for a supplement sample and could detect the d-CAR contamination at the 2.5% level, suggesting its potential for use in the purity test of CAR preparations.

Chinese hamster ovary (CHO) cell expression systems are widely utilized for biologics manufacturing due to their efficient recombinant protein expression and human-like post-translational modifications. With an increasing demand for bio-therapeutic proteins, the development of high-yielding CHO cell lines and refining bioprocess parameters are key to achieving reliable, efficient, and cost-effective therapeutic protein production. Many lipidomic studies on CHO cells have highlighted the crucial roles of lipids in supporting cell growth and specific productivity, offering valuable insights for optimizing culture media and metabolic pathways. Among these lipid classes, sterols such as cholesterol and its downstream intermediates have been investigated in CHO cells, though their functions remain less thoroughly explored compared to overall lipid metabolism. In this study, comprehensive lipidomic profiling, including cholesterol and esterified cholesterol, was performed on two distinct basal media and feed conditions, which identified lipid composition changes that offer mechanistic insights into how culture conditions potentially influence membrane dynamics. These findings provide a foundation for future development of culture media and bioprocess control strategies that can ultimately improve yield and process consistency.

The formation of M^+∙, [M − 2H]^+∙, and [M − nH]⁺ (n = 1, 3, 5) ions has been reported by using atmospheric pressure corona discharge (APCD) of n-heptane. The M^+∙ and [M − H]⁺ ions can be produced by high-electric field tunnel ionization (HEFTI) and hydride abstraction reactions, respectively. It has been proposed that the [M − 2H]^+∙, [M − 3H]⁺, and [M − 5H]⁺ ions are produced by the loss of hydrogen atoms from the [M − H]⁺ ion. The geometric and electronic configurations of the M^+∙, [M − 2H]^+∙, and [M − nH]⁺ (n = 1, 3, 5) ions were determined by quantum chemical calculations (QCCs). The product ions resulting from higher-energy collisional dissociation (HCD) of the M^+∙ ion can be characterized by the loss of alkyl radicals and alkanes, while those of the [M − 2H]^+∙ ion can be characterized by the loss of alkyl radicals and alkenes. The HCD product ions of the [M − nH]⁺ (n = 1, 3, 5) ions can be fundamentally characterized by the loss of alkenes. The product ions present in the HCD spectra can be supported by the geometric and electronic configurations determined by the QCCs. The fragment ions observed in the APCD mass spectrum are likely attributable to the result of collisional interactions between the M^+· ion and the air components such as N₂, O₂, and Ar, or to the result of the HEF-induced cracking reaction leading to the alkyl fragments.

The precise control of specific protein expression in target cells remains a critical challenge for the advancement of messenger RNA (mRNA) therapeutics. While incorporating regulatory sequences, such as microRNA-responsive RNA switches, is a promising approach for cell-type-specific targeting, optimizing these sequences requires extensive and time-consuming screening using conventional evaluation methods. In this study, we applied a multiplexed evaluation methodology utilizing peptide barcodes coupled with liquid chromatography-mass spectrometry (LC-MS) to improve the screening and design efficiency of mRNA expression control sequences. We designed mRNAs equipped with cell-specific expression control sequences and fused unique peptide barcodes to the encoded target proteins. By comparing conventional individual transfections with simultaneous co-transfections, we demonstrated that our LC-MS-based method can successfully distinguish and quantify protein expression derived from individual mRNA candidates within a single mixed sample. The simultaneous evaluation using peptide barcodes yielded results equivalent to the individual evaluations but required a reduced number of samples. This multiplexed technology would accelerate the screening process for regulatory sequences and is expected to enhance the development of mRNA therapeutics with high targeting specificity.

Human induced pluripotent stem cell (iPSC)-derived neural cells (NCs) and cardiomyocytes (CMs) show significant promise for clinical applications in regenerative medicine. Because the manufacturing of iPSC-derived products faces challenges in quality reproducibility, cost, and manufacturing time, closed automated culture systems have been developed. Recently, an efficient and noninvasive cell monitoring system based on extracellular markers secreted by cells represents a new strategy for closed systems. Extracellular vesicles (EVs), secreted by cells, contain proteins involved in stem cell differentiation; however, EV proteins in the cell culture medium are difficult to analyze because of their low abundance. Thus, their changes during cell differentiation, and the relationship between EV and cell proteins, remain unclear. In this study, we investigated the changes in EV proteins during iPSC differentiation into NCs and CMs using data-independent acquisition LC/MS/MS (DIA-MS), a highly comprehensive and quantitative approach. We observed significant positive correlations between the EV protein abundance and cellular protein expression levels during differentiation on days 0–7, and identified signature proteins characteristic of NCs and CMs. Among these, trophoblast glycoprotein (TPBG) for NC differentiation and keratin, type I cytoskeletal 19 (KRT19) for CM differentiation were particularly increased in both cells and EVs. Moreover, TPBG and KRT19 levels at day 7 showed similar trends to the differences in differentiation observed among the cell lines, as determined by flow cytometry. These findings suggest that these EV proteins may serve as predictive markers for monitoring differentiation into NC and CM.

Instrumental dispersion in the ion source can severely distort fast chromatographic peaks in supercritical fluid chromatography (SFC)–mass spectrometry (MS). Despite this importance, the dispersion characteristics specific to medium-vacuum chemical ionization (MVCI) sources have not been quantitatively investigated. In this work, we combine targeted experiments with established computational tools—computational fluid dynamics (CFD) and electrostatic field simulation—to characterize ion transport in the MVCI flow tube. Arrival profiles of vitamin K₁ (VK₁) ions monitored by selected ion monitoring consistently showed a reproducible two-component structure consisting of an early narrow bandwidth ion packet followed by a delayed shoulder. CFD calculations reproduce this tailing peak profile, and electrostatic modeling further revealed that applying the same potential to the MVCI flow tube and inner cylinder generates lateral potential walls that inhibit long-residence-time ions from entering the skimmer. Introducing an appropriate potential difference between the MVCI inner cylinder and the skimmer orifice isolates the MVCI flow field from the ion guide region and suppresses long residence-time trajectories, which narrows the VK₁ peak width by more than threefold and restores the intrinsic column efficiency of a sub-2-μm SFC column (from the theoretical plate (N) = 3120 to 12079). These results provide a practical diagnostic and mitigation framework for ion-source derived dispersion in MVCI, demonstrating that modest electrostatic confinement is effective in maintaining chromatographic fidelity in high-speed SFC–MVCI–MS. The present work also highlights the utility of CFD for evaluating proposed MVCI flow designs and their influence on peak dispersion.

The stop-and-go extraction tip (StageTip) is widely used for peptide purification in bottom-up proteomics, yet the original 3M Empore disk is no longer available, prompting the need to evaluate current alternatives. Two types of commercially available poly(styrene-divinylbenzene) (SDB)-containing polytetrafluoroethylene (PTFE) disks were used to fabricate StageTips. Desalting was performed on 500 and 20 ng of HeLa tryptic peptides, and the nanoscale liquid chromatography-tandem mass spectrometry results were compared. Both StageTips demonstrated equivalent performance for the 500 ng sample, but for the 20 ng sample, one StageTip identified 1.8 times more peptides, particularly longer and more hydrophobic peptides. Physical characterization and scanning electron microscope imaging of these disks revealed that the high-performing disk contains more PTFE fibers, partially covering the SDB particle surface. This likely prevents hydrophobic peptides from being trapped in small mesopores, improving recovery from low-input samples. These findings demonstrate that disk material properties critically influence performance in trace proteomics.

In previous work, a flash desorption/rapid-cooling system for the analysis of nonvolatile samples was developed using a heated needle. The needle was moved down to the sample surface using a linear actuator. In this system, it was necessary to adjust the lowest position of the needle manually. In the present work, a flash-heating system that requires no manual adjustment was implemented and combined with an alternating current (AC) corona discharge ion source. By using a movable needle inserted into a heated ceramic tube, the needle stops and remains on the sample surface at the moment the needle tip touches it, thereby preventing deep penetration. After contact for 200 ms, the needle was lifted up, enabling rapid cooling of the heated sample. Thus, the movable needle in the ceramic tube acts as a soft-touch sensor. This system, coupled with an AC corona discharge ion source, was successfully applied to the analysis of industrial polymers.

Native mass spectrometry (MS) enables the direct observation of non-covalent protein interactions while preserving the higher-order structures of protein complexes. We previously developed direct native MS approaches for analyzing overexpressed protein complexes from crude lysates and single human cells, retaining their interactions with small molecules and drugs. Here, we demonstrate that this strategy can be extended to endogenous protein complexes, which are present at substantially lower abundance than the overexpressed protein complexes analyzed in previous direct native MS studies, by directly analyzing crude lysates of cultured human HEK293T cells without prior purification or buffer exchange. Although crude human cell lysates contain salts and matrix compounds that typically cause peak broadening and signal suppression, we successfully observed the endogenous ~145-kDa glyceraldehyde-3-phosphate dehydrogenase (GAPDH) tetramer directly from unpurified human cell lysates, including its NAD⁺-bound forms. These results demonstrate the feasibility of directly detecting an endogenous high-molecular-weight protein complex and its cofactor-binding states from crude human cultured cell lysates, providing a basis for extending this approach to other endogenous protein assemblies and future single-cell native MS analyses.