Type 2 diabetes mellitus is a global health problem, placing patients at a higher risk of developing cardiovascular diseases and cancer. This study investigates the antidiabetic potential of Cassia bakeriana bark extracts and fractions. We evaluate their ability to inhibit α-amylase and α-glucosidase enzymes and advanced glycation end-products (AGEs). The antioxidant potential was also examined. Extracts were prepared through maceration with hexane (HE) and ethanol (EE), and the fractions were obtained via liquid-liquid extraction from EE. Anti-enzymatic, anti-glycation, antioxidant, and cytotoxic assays were conducted in 96-well plates using different concentrations of samples to determine the half-maximal inhibitory concentration (IC). Active samples were further analyzed using HPLC-(-)-ESI-MS/MS. The ethyl acetate fraction (EAF) demonstrated a high percentage of α-amylase inhibition (94.0%) with a promising IC value of 1.05 μg mL. Additionally, EAF displayed 61.5% inhibition of α-glucosidase, with an IC value of 537 μg mL. The EE, EAF, and n-butanol fraction (BF) exhibited strong anti-glycation capacities. Furthermore, the EE, EAF, BF, and dichloromethane fractions showed promising antioxidant activity using the DPPH and ORAC methodologies. Cytotoxic activity was also evaluated with Vero cells, revealing no adverse effects on cell viability (CC > 512.0 μg mL). Active samples predominantly comprised proanthocyanidins, flavonoids, and anthraquinone, representing the main constituents of C. bakeriana bark. This study provides the first assessment of the antidiabetic potential of C. bakeriana bark and a comprehensive analysis of the chemical composition of its active extracts and fractions, offering hope for future treatments.

The spatial information of xenobiotics distribution, metabolism, and toxicity mechanisms in situ has drawn increasing attention in both pharmaceutical and environmental toxicology research to aid drug development and environmental risk assessments. Mass spectrometry imaging (MSI) provides a label-free, multiplexed, and high-throughput tool to characterize xenobiotics, their metabolites, and endogenous molecules in situ with spatial resolution, providing knowledge on spatially resolved absorption, distribution, metabolism, excretion, and toxicity on the molecular level. In this perspective, we briefly summarize applications of MSI in toxicology on xenobiotic distribution and metabolism, quantification, toxicity mechanisms, and biomarker discovery. We identified several challenges regarding how we can fully harness the power of MSI in both fundamental toxicology research and regulatory practices. First, how can we increase the coverage, sensitivity, and specificity in detecting xenobiotics and their metabolites in complex biological matrices? Second, how can we link the spatial molecular information of xenobiotics to toxicity consequences to understand toxicity mechanisms, predict exposure outcomes, and aid biomarker discovery? Finally, how can we standardize the MSI experiment and data analysis workflow to provide robust conclusions for regulation and drug development? With these questions in mind, we provide our perspectives on the future directions of MSI as a promising tool in spatial toxicology research.

To discriminate amino acid isomers by multiple stage tandem mass spectrometry (MS), the fragmentation of protonated amino acids were investigated by MS with collision-induced dissociation (CID) and density functional theory calculations. The CID of protonated α-amino acids results in a loss of 46 Da, corresponding to HO and CO, and iminium ions appear as resultant fragments. The CID of protonated β-amino acids also produces iminium ions, but the corresponding loss is 60 Da instead of 46 Da. HO loss initiates the fragmentation of protonated β-amino acids, producing protonated β-lactams as an intermediate. Subsequently, protonated β-lactams are easily converted to iminium ions and CHCO. By contrast, HO loss from the protonated forms of γ- and ε-amino acids provides protonated lactams with 5- and 7-membered rings, respectively. Protonated lactams with more than 5-membered rings provide stable fragments and do not undergo further degradation during CID. In addition, protonated forms of γ- and ε-amino acids undergo NH loss as a competitive fragmentation pathway of HO loss, producing protonated lactones. Because the fragmentation of protonated amino acid by CID depends on the position of amino and carboxyl groups, the tandem mass spectrometry with CID can discriminate α-, β-, and other amino acids.

A novel inorganic nonmetallic material boron carbide (BC) was applied in paper spray ionization to analyze organic molecules in both negative and positive modes. The utilization of BC has led to a notable enhancement in signal responses for various molecules, including bisphenols and drugs, by approximately two to four times. The limit of detection (LOD) of bisphenol AF and nilotinib standard solutions can reach 1 and 0.5 μg/mL, respectively. Moreover, linear relationships for bisphenol AF was established within the concentration range of 1-100 μg/mL, exhibiting strong correlation coefficients. Recovery experiments for BPAF in water samples from the inlet and outlet of a sewage treatment plant, conducted without any pretreatment, achieved a maximum recovery rate of 98.8% and an RSD below 9.78%.

Hydrogen deuterium-exchange mass spectrometry (HDX-MS) is commonly used in the study of protein dynamics and protein interactions. By measuring the isotopic exchange of backbone amide hydrogens in solution, HDX-MS offers valuable structural insights into challenging biological systems. Traditional HDX-MS approaches utilize bottom-up (BU) proteomics, in which deuterated proteins are digested before MS analysis. BU-HDX enables the characterization of proteins with various sizes in simple protein mixtures or complex biological samples such as cell lysates. However, BU methods are inherently limited by the inability to resolve protein sub-populations arising from different protein conformations, such as those arising from post-translational modifications (PTMs). Alternatively, top-down (TD) HDX-MS detects the global deuterium uptake at the intact proteoform level, allowing direct probing of structural changes due to protein-protein interactions, PTMs, or conformational changes. Combining TD-HDX-MS with electron-based fragmentation techniques, such as electron capture dissociation (ECD) and electron transfer dissociation (ETD), has demonstrated the feasibility of studying intact protein interactions with amino acid-level resolution. Here, we present a brief overview of methodologies, limitations, and applications of TD-HDX-MS using direct infusion techniques and LC-based approaches. Furthermore, we conclude with a perspective on the future directions for TD-HDX-MS.

Protein structure, including various post-translational modifications and higher-order structures, regulates diverse biological functions. Native mass spectrometry (native MS) is a powerful analytical technique used to determine the masses of biomolecules, such as proteins and their complexes, while preserving their native folding in solution. This method provides structural information on the composition of monomers or complexes and the stoichiometry of subunits within each complex, significantly contributing to protein structural analysis. Native MS has evolved to incorporate top-down approaches, enabling the characterization of proteoforms and non-covalent interactions between metabolites or proteins and specific targets. This perspective highlights the advancements in native MS for intracellular proteins and protein complexes, and discusses future research directions toward cellular biology.

Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) uses an infrared laser to desorb neutral biomolecules with postionization via ESI at atmospheric pressure. The Gaussian profile of the laser with conventional optics results in the heating of adjacent nonablated tissue due to the energy profile being circular. A diffractive optical element (DOE) was incorporated into the optical train to correct for this disadvantage. The DOE produces a top-hat beam profile and square ablation spots, which have uniform energy distributions. Although beneficial to mass spectrometry imaging (MSI), it is unknown how the DOE affects the ability to perform quantitative MSI (qMSI). In this work, we evaluate the performance of the DOE optical train against our conventional optics to define the potential advantages of the top-hat beam profile. Absolute quantification of glutathione (GSH) was achieved by normalizing the analyte of interest to homoglutathione (hGSH), spotting a dilution series of stable isotope labeled glutathione (SIL-GSH), and analyzing by IR-MALDESI MSI with either the conventional optical train or with the DOE incorporated. Statistical comparison indicates that there was no significant difference between the quantification of GSH by the two optical trains as evidenced by similar calibration curves. Results support that both optical trains can be used for qMSI without a change in the ability to carry out absolute quantification but providing the benefits of the top-hat optical train (i.e., flat energy profile and square ablation spots)-for future qMSI studies.

The leading type of ionization technique in mass spectrometry analyses is the ionization at atmospheric pressure. The aim of this study was to assess ESI and APCI ionization efficiency of pesticides introduced to the MS source in four organic phases, non-doped and doped with formic acid and ammonium formate. Ionization efficiency in modified ESI and APCI, applying in-source sample heating, was also assessed. The study was primarily designed to support non-chromatography-based mass spectrometry pesticides analysis by the direct infusion technique. Evaluation of analysis performances such as calibration performances, detectability, and sensitivity should indicate a top solvent-additive-source combination, leading to the highest ionization efficiency and lowest analytes detection limits.

A peptide's fractional mass is directly associated with its elemental composition and thus amino acid composition. Here it is demonstrated that a peptide's fractional mass alone can be a useful identifier or indicator of that composition for small to mid-sized peptides (5-7 amino acids) and can significantly reduce the number of viable amino acid compositions for larger peptides (> 8 residues) to include or exclude certain possibilities. Separate consideration of the integer portion of the peptide's mass helps to reduce the number of possibilities where many duplicate fractional mass values are found. Adoption of this fractional mass strategy should aid approaches that are presently employed for peptide identification, including in the use of mass map data to search protein databases for proteomics applications.

Atmospheric pressure chemical ionization (APCI) is often used in the analysis of linear saturated hydrocarbons (LSHs) as this ionization technique commonly produces [M - H] ions in high abundance. However, APCI (along with other atmospheric pressure sources) is often impacted by in-source oxidation, leading to a variety of ionic products. Identifying these products and understanding their mechanisms of formation is crucial for characterizing complex mixtures with substantial hydrocarbon content, such as those found in the petrochemical industry. In this study, in-source oxidation of LSHs was observed in gas chromatography (GC) coupled to high-resolution mass spectrometry (HRMS) via a custom-built APCI interface. Studies showed that the abundance of these oxidized ions correlated positively with atmospheric water, yet occurred without the inclusion of water-based oxygen as judged by experiments with stable isotope-labeled water. The oxidation of LSHs was further influenced by the reactive species in the ionization atmosphere. Fragmentation data using stable isotope-labeled LSH standards unveiled multiple structurally unique ions with one or more oxidation sites on both primary and secondary carbons. These ionic products bear resemblance to combustion byproducts, suggesting the instrumental configuration fosters plasma-assisted combustion-like processes that encourage the radical-mediated oxidation of LSHs rather than generate [M - H]. Through these investigative efforts, a mechanism analogous to combustion was proposed for the formation of LSH oxidation products in GC-APCI-HRMS. Data demonstrate that these ions are robustly generated in petrochemical products, allowing for proper characterization of these complex mixtures.

The estimation of relative levels of amino acids is crucial for understanding various biological processes in plants, including photosynthesis, stress tolerance, and the uptake and translocation of nutrients. A wide range of liquid chromatography (LC; HPLC/UHPLC)-based methods is available for measuring the quantity of amino acids in plants. Additionally, the coupling of LC with mass spectrometry (MS) significantly enhanced the robustness of existing chromatographic methods used for amino acid quantification. However, accurate annotation and integration of mass peaks can be challenging for plant biologists with limited experience in analyzing MS data, especially in studies involving large datasets with multiple treatments and/or replicates. Further, there are instances when the experiment demands an overall view of the amino acids profile rather than focusing on absolute quantification. The present protocol provides a detailed LC-MS method for obtaining a qualitative amino acids profile using MS-DIAL, a versatile and user-friendly program for processing MS data. Free amino acids were extracted from the leaves of control and Tomato leaf curl Palampur virus (ToLCPalV)-infected Nicotiana benthamiana plants. Extracted amino acids were derivatized and separated using UHPLC-QTOF, with each amino acid subsequently identified by aligning mass data with a custom text library created in MS-DIAL. Further, MS-DIAL was employed for internal standard-based normalization to obtain a qualitative profile of 15 amino acids in control and virus-infected plants. The outlined method aims to simplify the processing of MS data to quickly assess any modulation in amino acid levels in plants with a higher degree of confidence.

A novel method was developed for the rapid detection of heavy metals in firework solutions with high sensitivity and minimal pretreatment by enriching them with ethylenediaminetetraacetic acid (EDTA) reagent and analyzing them using microwave plasma torch mass spectrometry (MPT-MS). Quantitative results showed that the limits of detection and quantification for heavy metals (Pb, Ba, Sr, and Ag) ranged from 0.05 to 0.25 and 0.38 to 0.71 μg·L, respectively. The linear dynamic ranges covered at least two orders of magnitude, with correlation coefficients exceeding 0.99. Fireworks from five regions in China were also analyzed quantitatively, detecting heavy metals including Pb, Ba, Sr, and Ag, with recovery rates ranging from 87.9% to 107.5%. Good separation between the firework samples from different regions was achieved by using element ratios and principal component analysis (PCA). These results from the preliminary study showed that the EDTA-assisted MPT-MS combined with PCA is a powerful tool for characterizing firework samples and tracing them back to their sources, which is valuable to effectively regulate and manage banned fireworks.

Bacterial keratitis (BK) is an infection that causes inflammation of the cornea and, if severe, can result in blindness. Topical fluoroquinolones combined with corticosteroids have been shown to be useful in the treatment of BK. A rapid, selective, and sensitive bioanalytical method for simultaneous quantification of Gatifloxacin (GAT) and Dexamethasone (DEX) has been developed and validated using tandem mass spectrometry (LC-MS/MS). Optimal separation was accomplished in under 5 min using an Agilent Zorbax C18 column (100 mm × 4.6 mm, 3.5 μm). The mobile phase was composed of a blend of 0.2% formic acid in triple distilled water and methanol with a flow rate of 0.65 mL/min in isocratic mode. GAT and DEX were detected in positive electrospray ionization multiple reaction monitoring mode (MRM), and the retention time was found to be at 1.64 and 2.93 min, respectively. The linearity of GAT and DEX was found to be in the range of 1.56-400 ng mL with good precision and accuracy. The method was validated according to USFDA regulatory guidelines. The validated method was effectively utilized for preclinical pharmacokinetic analysis of GAT and DEX in rabbit tear fluid following the topical application of a commercial formulation.

The unimolecular reactions of protonated myrcene and linalool were investigated by collision-induced dissociation and density functional theory calculations. Experiments on a triple quadrupole mass spectrometer showed that protonated myrcene undergoes two major unimolecular reactions losing propene and isobutene, and two minor reactions of ethene and propane loss. In each case, the product ion consists of a substituted five-member ring. Protonation of myrcene was found to form four distinct protomers, three of which can be significantly populated in the ion source. The observed fragmentation reactions were calculated and found to depend on the starting protomer. Each pathway consisted of several hydrogen-migration and ring-forming/opening steps on the way to the observed products. Likewise, protonation of linalool also produces three distinct protomers, with the global minimum being formed by protonation of a central double bond. The major reaction is water loss to form protonated myrcene, but two minor channels were also observed resulting in loss of acetone and isobutene. The calculated minimum energy reaction pathways were found to be consistent with the relative abundances of the ions in the experimental breakdown diagrams.

This study employs a high-dimensional consensus mass spectral (HDCMS) similarity scoring technique to discriminate isomers collected using an electron ionization mass spectrometer. The HDCMS method was previously introduced and applied to the discrimination of mass spectra of constitutional isomers, methamphetamine and phentermine, collected with direct analysis real-time mass spectrometry (DART-MS). The method formulates the problem of discriminating mass spectra in a mathematical Hilbert space and is hence called "high dimensional." It requires replicate mass spectra to build a Gaussian model and evaluate the inner products between these functions. The resulting measurement variability is used as a signature by which to discriminate spectra. In this work, HDCMS is tested on electron impact ionization (EI) mass spectra of 7 terpene and terpene-related (CH and CH) isomers with experimental retention indices that differ by less than 30 and with traditional cosine similarity scores greater than 0.9, on a scale of 0 to 1, when compared with at least one other compound in the test set. Using identical instrument parameters, 15 replicate gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) spectra of each isomer were collected and separated into distinct library and query sets. The HDCMS algorithm discriminated each isomer, indicating the method's potential. Because the method requires replicate measurements, observations from a simple heuristic study of the number of replicates required to discriminate these isomers is presented. The paper concludes with a discussion of compound discrimination using HDCMS and the benefits and drawbacks of applying the method to EI-MS data.

The study of metal ion's role in the biological processes of Alzheimer's disease has spurred investigations into the coordination chemistry of amyloid beta peptide and its fragments. Nano-electrospray ionization mass spectrometry (nESI-MS) has been utilized to examine the stabilization of bound anions on multiprotein complexes without bulk solvent. However, the effects of anions on metal ion binding interactions with amyloid beta peptide have not been explored. This study directly examined metal-peptide complexes using nESI-MS and investigated the effects of various anions on the binding ratio and stability of these complexes from ammonium salt solutions. The results indicate that different anions have distinct effects on the binding ratio and stability of various metal-peptide complexes. Of these, the bicarbonate ion exhibits the highest binding ratios for metal-peptide complexes, while binding ratios for these complexes in phosphate are comparatively low. Our results suggest that acetate, formate, bicarbonate, and phosphate have weak affinities and act as weak stabilizers of the metal-peptide complex structure in the gas phase. Intriguingly, chloride and sulfate act as stabilizers of the metal-peptide complex in the gas phase. The rank order determined from these data is substantially different from the Hofmeister salt series in solution. Although this outcome was anticipated due to the reduced influence of anions and water solvation, our findings correlate well with expected anion binding in solution and emphasize the importance of both hydration layer and anion-metal-peptide binding effects for Hofmeister-type stabilization in solution. This approach proved useful in examining the interactions between metal ions and amyloid beta peptide, which are relevant to Alzheimer's disease, using direct ESI-MS.

Protein glycosylation is the co- and/or post-translational modification of proteins with oligosaccharides (glycans). This process is not template based and can introduce a heterogeneous set of glycan modifications onto substrate proteins. Glycan structures preserve biomolecular information from the cell, with glycoproteins from different cell types and tissues displaying distinct patterns of glycosylation. Several decades of research have revealed that glycan structures also differ between normal physiology and disease. This suggests that the information stored in glycoproteins and glycans can be utilized for disease diagnosis and monitoring. Methods that enable sensitive and site-specific measurement of protein glycosylation in clinical settings, such as nano-flow liquid chromatography tandem mass spectrometry, are therefore essential. The purpose of this perspective is to discuss recent advances in mass spectrometry and the potential of these advances to facilitate the detection and monitoring of disease-specific glycoprotein glycoforms. Glycoproteomics, the system-wide characterization of glycoprotein identity inclusive of site-specific characterization of carbohydrate modifications on proteins, and glycomics, the characterization of glycan structures, will be discussed in this context. Quantitative measurement of glycopeptide markers via parallel reaction monitoring is highlighted. The development of promising glycopeptide markers for autoimmune disease, liver disease, and liver cancer is discussed. Synthetic glycopeptide standards, ambient ionization mass spectrometry, and consideration of glyco-biomarkers in two- and three-dimensional space within tissue will be critical to the advancement of this field. The authors envision a future in which glycoprotein mass spectrometry workflows will be integrated into clinical settings, to aid in the rapid diagnosis and monitoring of disease.

We evaluated the performance of Zybio EXS2600 matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Zybio Inc., Chongqing, China) for the identification of bacteria from positive blood culture (BC) bottles using Blood Culture Positive Sample Pretreatment Kit (Zybio Inc., Chongqing, China) in comparison to an in-house saponin method. Following a positive signal by the BACTEC™ FX system, confirmation of identification was achieved using subcultured growing biomass used for MALDI-TOF MS analysis. A total of 94 positive BC bottles with 97 bacterial isolates were analyzed. The overall identification rates at the genus and species levels for the saponin method were 89.7% (87/97) and 74.2% (72/97), respectively. With the Zybio Kit, 88.7% (86/97) and 80.4% (78/97) of microorganisms were correctly identified to the genus and species levels, respectively. The saponin method identified 65.3% (32/49) of Gram-positive bacteria at the species level, whereas the Zybio Kit achieved a higher species-level identification rate of 79.6% (39/49) (p = 0.1153). The saponin method with additional on-plate formic acid extraction showed a significantly higher overall identification rate in comparison to the saponin method without that step for both genus (87.6% [85/97] vs. 70.1% [68/97], p = 0.0029) and species level (70.1% [68/97] vs. 46.4% [45/97], p = 0.0008). Identification rates of Gram-negative bacteria showed a higher identification rate, however, not statistically significant with additional Zybio Kit protocol step on both genus (85.4% [41/48] vs. 81.3% [39/48], p = 0.5858) and species level (77.1% [37/48] vs. 75% [36/48], p = 0.8120). Zybio Kit could offer an advantage in species-level identification, particularly for Gram-positive bacteria. The inclusion of on-plate formic acid extraction in the saponin method notably enhanced identification at both genus and species levels for Gram-positive bacteria. The extended protocol provided by the Zybio Kit could potentially offer an advantage in the identification of Gram-negative bacteria at both genus and species levels. Enhancements to the Zybio EXS2600 MALDI-TOF instrument software database are necessary.

Apixaban, a direct oral anticoagulant drug (DOAC), typically does not require routine therapeutic drug monitoring (TDM), yet recent guidelines propose its use in specific clinical scenarios. While various antifactor Xa (anti-FXa) chromogenic assays serve as useful proxies for measuring plasma exposure to apixaban in emergencies, they lack specificity compared with chromatographic methods. This research project is intended to the development and validation of a standardized protocol of liquid chromatography-tandem mass spectrometry (LC-MS/MS) in conformity with the ICH guidelines M10 for the measurement of apixaban in both plasma and dried plasma spots (DPSs). Samples preparation included protein precipitation after the addition of a deuterated internal standard (IS), and the chromatographic separation was carried out on a Thermo Scientific™ Accucore™ Polar Premium column (50 mm × 2.1 mm, i.d. 2.6 m). The newly developed LC-MS/MS method for apixaban mesurement from both plasma and DPS resulted linear over a wide concentration range (31.25-500 ng/mL), accurate, and reproducible without matrix effects, allowing for specific and rapid quantification. Stability was assessed on quality controls and a real sample, allowing the setting up of a robust TDM protocol that was applied to five anonymized plasma samples obtained from adult patients undergoing apixaban treatment at steady-state. In conclusion our novel LC-MS/MS method is adequate for accurate apixaban quantitation from both plasma and DPS matrixes, and may thus facilitate the guidelines suggested implementation of apixaban TDM, even in peripheral hospitals through shipment of DPS at reference laboratories.

Clinical expression of coronavirus disease 2019 (COVID-19) infectionis widely variable including fatal cases and patients with mild symptoms and a rapid resolution. We studied saliva from 63 hospitalized COVID-19 patients and from 30 healthy controls by integrating large-scale proteomics, peptidomics and targeted metabolomics to assess the biochemical alterations following the infection and to obtain a set of putative biomarkers useful for noninvasive diagnosis. We used an untargeted approach by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for proteomics and peptidomics analysis and targeted LC-multiple reaction monitoring/MS for the analysis of amino acids. The levels of 77 proteins were significantly different in COVID-19 patients. Among these, seven proteins were found only in saliva from patients with COVID-19, four were up-regulated and three were down-regulated at least five-folds in saliva from COVID-19 patients in comparison to controls. The analysis of proteins revealed a complex balance between pro-inflammatory and anti-inflammatory proteins and a reduced amount of several proteins with immune activity that possibly favours the spreading of the virus. Such reduction could be related to the enhanced activity of endopeptidases induced by the infection that in turn caused an altered balance of free peptides. In fact, on a total of 28 peptides, 22 (80%) were differently expressed in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and control subjects. The multivariate analysis of such peptides permits to obtain a diagnostic algorithm that discriminate the two populations with a high diagnostic efficiency. Among amino acids, only threonine resulted significantly different between COVID-19 patients and controls, while alanine levels were significantly different between COVID-19 patients with different severity. In conclusion, the present study defined a set of molecules to be detected with a quick and easy method based on mass spectrometry tandem useful to reveal biochemical alterations involved in the pathogenesis of such a complex disease. Data are available via ProteomeXchange with identifier PXD045612.