[Identification and visual analysis of ginsenosides in multi-steamed roots of Panax quinquefolium based on UPLC-Q-TOF-MS/MS and MALDI-MSI].

This study aims to investigate the component variations and spatial distribution of ginsenosides in Panax quinquefolium roots during repeated steaming and drying. Ultra performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry(UPLC-Q-TOF-MS/MS) was employed to identify the ginsenosides in the root extract. Matrix-assisted laser desorption/ionization mass spectrometry imaging(MALDI-MSI) was employed to visualize the spatial distribution and spatiotemporal changes of prototype ginsenosides and metabolites in P. quinquefolium roots. The UPLC results showed that 90 ginsenosides were identified during the steaming process of the roots, and polar ginsenosides were converted into low polar or non-polar ginsenosides. The content of prototype ginsenosides decreased, while that of rare ginsenosides increased, which included 20(S/R)-ginsenoside Rg_3, 20(S/R)-ginsenoside Rh_2, and ginsenosides Rk_1, Rg_5, Rs_5, and Rs_4. MALDI-MSI results showed that ginsenosides were mainly distributed in the epidermis and phloem. As the steaming times increased, ginsenosides were transported to the xylem and medulla. This study provides fundamental information for revealing the changes of biological activity and pharmacological effect of P. quinquefolium roots that are caused by repeated steaming and drying and gives a reference for expanding the application scope of this herbal medicine.

Nutrient supplementation-induced metabolic profile changes and early appearance of free N-glycans in nutrient deficient tomato plants revealed by mass spectrometry.

Inorganic fertilizers are routinely used in large scale crop production for the supplementation of nitrogen, phosphorus, and potassium in nutrient poor soil. To explore metabolic changes in tomato plants grown on humic sand under different nutritional conditions, matrix-assisted laser desorption ionization (MALDI) mass spectrometry was utilized for the analysis of xylem sap. Variations in the abundances of metabolites and oligosaccharides, including free N-glycans (FNGs), were determined. Statistical analysis of the sample-related peaks revealed significant differences in the abundance ratios of multiple metabolites, including oligosaccharides, between the control plants, grown with no fertilizers, and plants raised under "ideal" and "nitrogen deficient" nutritional conditions, i.e., under the three treatment types. Among the 36 spectral features tentatively identified as oligosaccharides, the potential molecular structures for 18 species were predicted based on their accurate masses and isotope distribution patterns. To find the spectral features that account for most of the differences between the spectra corresponding to the three different treatments, multivariate statistical analysis was carried out by orthogonal partial least squares-discriminant analysis (OPLS-DA). They included both FNGs and non-FNG compounds that can be considered as early indicators of nutrient deficiency. Our results reveal that the potential nutrient deficiency indicators can be expanded to other metabolites beyond FNGs. The m/z values for 20 spectral features with the highest variable influence on projection (VIP) scores were ranked in the order of their influence on the statistical model.

Clinical Application of Matrix-Assisted Laser-Resolved Ionization Time-Of-Flight Mass Spectrometry for Rapid Detection of Blood-Borne Pathogens.

Objective: To evaluate the performance of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in the identification of clinical pathogenic microorganisms. Methods: Blood culture-positive specimens were collected from inpatients in our hospital from March to December 2022 and identified using VITEK 2XL (biochemical), VITEK MS (colony), VITEK MS (bacterial membrane) and VITEK MS (separating gel) methods, respectively, to compare the compliance rate and identification values of the four methods. Results: A total of 280 strains were included in the analysis, including 155 (55.36%) Gram-negative and 125 (44.64%) Gram-positive strains. 279 (99.64%) of the 280 strains were identified by VITEK 2XL (biochemical), including 154 (99.35%) Gram-negative and 125 (100%) Gram-positive strains. VITEK MS (colony) identified 278 (99.29%) strains, including 153 (98.71%) Gram-negative and 125 (100%) Gram-positive. 261 (93.21%) strains were identified in VITEK MS (bacterial membrane), including 148 (95.48%) Gram-negative and 113 (90.40%) Gram-positive strains. VITEK MS (separating gel) identified 232 (82.86%) strains, including 136 (87.74%) Gram-negative and 96 (76.80%) Gram-positive strains. Conclusion: MALDI-TOF MS findings are highly consistent with traditional culture identification methods in terms of identification accuracy, and the VITEK MS (bacterial membrane) and VITEK MS (separating gel) identification methods significantly reduce the turnaround time for identification in the laboratory.

Imaging plant metabolism in situ.

Mass spectrometry imaging (MSI) has emerged as an invaluable analytical technique for investigating the spatial distribution of molecules within biological systems. In the realm of plant science, MSI is increasingly employed to explore metabolic processes across a wide array of plant tissues, including those in leaves, fruits, stems, roots, and seeds, spanning various plant systems such as model species, staple and energy crops, and medicinal plants. By generating spatial maps of metabolites, MSI has elucidated the distribution patterns of diverse metabolites and phytochemicals, encompassing lipids, carbohydrates, amino acids, organic acids, phenolics, terpenes, alkaloids, vitamins, pigments, and others, thereby providing insights into their metabolic pathways and functional roles. In this review, we present recent MSI studies that demonstrate the advances made in visualizing the plant spatial metabolome. Moreover, we emphasize the technical progress that enhances the identification and interpretation of spatial metabolite maps. Within a mere decade since the inception of plant MSI studies, this robust technology is poised to continue as a vital tool for tackling complex challenges in plant metabolism.

The visualization of the spatial distribution of Cocculus orbiculatus based on air flow-assisted desorption electrospray ionization mass spectrometry imaging.

Cocculus orbiculatus (C. orbiculatus), the root of plants belonging to the Menispermaceae family, has been extensively used to treat various diseases, including malaria and rheumatism. The main chemicals in these plants are alkaloids; however, the spatial distribution of these compounds within the plant roots remains undefined. This study aimed to visualize the spatial distribution of C. orbiculatus using air flow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI). In total, the spatial distribution of four aporphine alkaloids, five benzyltetrahydroisoquinoline alkaloids, six bisbenzylisoquinoline alkaloids, and one morphinane alkaloid in the cork layer, xylem, and ray of the root of C. orbiculatus was observed; the distribution characteristics of the different compounds in C. orbiculatus were significantly different. This study provides a visualized spatial distribution analysis method for the characterization of metabolites in the root tissue of C. orbiculatus and also provides valuable information for the specificity of the root of C. orbiculatus, which is beneficial for understanding its chemical separation, biosynthesis, and pharmacological activities.

State-of-the-art mass spectrometry imaging applications in biomedical research.

Mass spectrometry imaging has advanced from a niche technique to a widely applied spatial biology tool operating at the forefront of numerous fields, most notably making a significant impact in biomedical pharmacological research. The growth of the field has gone hand in hand with an increase in publications and usage of the technique by new laboratories, and consequently this has led to a shift from general MSI reviews to topic-specific reviews. Given this development, we see the need to recapitulate the strengths of MSI by providing a more holistic overview of state-of-the-art MSI studies to provide the new generation of researchers with an up-to-date reference framework. Here we review scientific advances for the six largest biomedical fields of MSI application (oncology, pharmacology, neurology, cardiovascular diseases, endocrinology, and rheumatology). These publications thereby give examples for at least one of the following categories: they provide novel mechanistic insights, use an exceptionally large cohort size, establish a workflow that has the potential to become a high-impact methodology, or are highly cited in their field. We finally have a look into new emerging fields and trends in MSI (immunology, microbiology, infectious diseases, and aging), as applied MSI is continuously broadening as a result of technological breakthroughs.

Laccase-Catalyzed Synthesis of Added-Value Polymers from Cork and Grape Extracts.

The development of products from natural plant sources, including agriculture and food wastes, contributes significantly to the circular economy and global sustainability. Cork and grape wastes were employed as the primary sources in this study to obtain compounds of interest under mild extraction conditions. Laccase was applied to oxidize the cork and grape extracts, with the aim of producing value-added molecules with improved properties. Ultraviolet-visible (UV-vis) spectroscopy was assessed to monitor the oxidation process, and characterization of the end products was performed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) spectroscopy. The antioxidant and antiaging properties were evaluated by means of ABTS, DPPH, FRAP, and SPF testing. Overall, as compared to their monomeric counterparts, the polymeric compounds displayed remarkable antioxidant and antiaging characteristics after laccase oxidation, showing tremendous potential for applications in the food, pharmaceutical, cosmetic, and textile industries.

Contribution of LDI and MALDI for the Characterization of a Lignocellulosic-Based Pyrolysis Bio-Oil.

In recent years, various alternatives to fossil fuels have been developed. One of them involves the production of bio-oils from lignocellulosic-based biomass through pyrolysis. However, bio-oils present numerous heteroatoms and, in particular, oxygen atoms that need to be removed by an upgrading process. To optimize these processes, it is necessary to have good knowledge of the composition of the bio-oils at the molecular level. This work aims to establish the usefulness of laser desorption ionization (LDI) and matrix-assisted laser desorption/ionization (MALDI) techniques on lignocellulosic biomass-based bio-oils. Using a Fourier transform ion cyclotron mass spectrometer (FTICR MS), we showed that MALDI gives more information than LDI. The selectivity of a series of MALDI matrices was investigated, showing that some matrices are selective toward compound families and others ionize a wider range of compounds. In this study, nine proton-transfer matrices and three electron-transfer matrices were used and compared to results obtained in LDI. Dithranol, acetosyringone, and graphene oxide were the three promising matrices selected from all matrices, giving an overall characterization of oxygenated classes in a bio-oil. They allowed the ionization of many more species covering a wide range of polarity, aromaticity, and mass with a homogeneous relative intensity for all molecular classes such as lignin-derivative species, sugars, and lipid-derivative species.

Stable isotope labeling as a promising tool for rapid drug susceptibility testing in Neisseria gonorrhoeae.

The world is heading towards an era of intractable and impending untreatable N. gonorrhoeae, thereby underlining the significance of rapid and accurate prediction of drug resistance as an indispensable need of the hour. In the present study, we optimized and evaluated a stable isotope labeling-based approach using the MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry) for rapid and reliable detection of ciprofloxacin and azithromycin resistance in N. gonorrhoeae. All the isolates were cultured under three varied condition setups viz. medium supplemented with normal lysine, heavy lysine (isotope), and heavy lysine along with the antibiotics (ciprofloxacin/azithromycin), respectively. After incubation, spectra were acquired using the MALDI-TOF MS which were further screened for unique patterns (media-specific spectra) to differentiate drug-susceptible and resistant isolates. The results of the stable isotope labeling assay were comparable to the results of phenotypic methods used for susceptibility testing.

Authentication of Single Herbal Powders Enabled by Microscopy-Guided In Situ Auto-sampling Combined with Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.

In the long history of investigation of herbal products, microscopic examination has greatly contributed to the authentication of herbs in a powder form. However, it cannot provide the chemical profiles of herbal powders and thus is limited to morphological identification. In this work, we present a label-free and automatic approach for the characterization and identification of single herbal powders and their adulterants, enabled through the combination of microscopy-guided auto-sampling and matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). To meet the demand for automatic and highly efficient in situ extraction, the glass slide was coated with gelatin to immobilize dried herbal powders that cannot stick to the glass slide like fresh and hydrated cells. The gelatin coating also facilitated the pump-out of chemical components and prevented diffusion across the interface enabled by the formation of a tight contact at the probe tip and surface. Optical microscopy was applied to acquire the microstructure and position of the herbal powders immobilized on the gelatin-coated slide. The candidate single herbal powders were picked out by a software for subsequent auto-sampling and MALDI MS identification. The combination of microstructure features and chemical profiles significantly improved the authentication capability of microscopic examination.

Identification of Radix Bupleuri From Different Geographic Origins Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry and Support Vector Machine Algorithm.

BACKGROUND: The geographic origin of Radix bupleuri is an important factor affecting its efficacy, which needs to be effectively identified. OBJECTIVE: The goal is to enrich and develop the intelligent recognition technology applicable to the identification of the origin of traditional Chinese medicine. METHOD: This article establishes an identification method of Radix bupleuri geographic origin based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and support vector machine (SVM) algorithm. The Euclidean distance method is used to measure the similarity between Radix bupleuri samples, and the quality control chart method is applied to quantitatively describe their quality fluctuation. RESULTS: It is found that the samples from the same origin are relatively similar and mainly fluctuate within the control limit, but the fluctuation range is large, and it is impossible to distinguish the samples from different origins. The SVM algorithm can effectively eliminate the impact of intensity fluctuations and huge data dimensions by combining the normalization of MALDI-TOF MS data and the dimensionality reduction of principal components, and finally achieve efficient identification of the origin of Radix bupleuri, with an average recognition rate of 98.5%. CONCLUSIONS: This newly established approach for identification of the geographic origin of Radix bupleuri has been realized, and it has the advantages of objectivity and intelligence, which can be used as a reference for other medical and food-related research. HIGHLIGHTS: A new intelligent recognition method of medicinal material origin based on MALDI-TOF MS and SVM has been established.

A novel visualization method for the composition analysis of processed garlic by MALDI-TOF imaging mass spectrometry (MSI) and Q-TOF LC-MS/MS.

Laba garlic is a kind of vinegar processed garlic (Allium sativum L.) product with multiple health effects. This study applied matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-TOF MSI) and Q-TOF LC-MS/MS for the first time to investigate the garlic tissue spatial distribution changes of low molecular weight compounds during the Laba garlic processing. The distribution characteristics of the compounds were observed in processed and unprocessed garlic including amino acids and derivatives, organosulfur compounds, pigment precursors, polysaccharides and saponins. During Laba garlic processing, some bioactive compounds such as alliin and saponins were lost because they were transformed into other compounds or leached into the acetic acid solution, and some new compounds including pigments-related compounds occurred. This study provided a basis for the spatial distributions and changes of compounds in garlic tissue during Laba garlic processing, which suggested that the bioactivities of garlic might be changed after processing owing to the transformation and change of the constituents.

Study on the spatial distribution of coumarins in Angelica dahurica root by MALDI-TOF-MSI.

INTRODUCTION: The main chemical components of Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav. are coumarins and volatile oils, and coumarins are regarded as the representative constituents with various pharmacological effects. OBJECTIVE: Based on matrix-assisted laser desorption/ionization time of flight mass spectrometry imaging (MALDI-TOF-MSI), a method for spatial distribution analysis of coumarins in primary root and lateral root of A. dahurica was established. Also, spatial visualization of coumarins in the roots of A. dahurica was realized. MATERIALS AND METHODS: α-Cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid, and 9-aminoacridine were used as matrices. MALDI-TOF-MSI was employed to analyze the standards of imperatorin, oxypeucedanin, and osthole. Based on the higher sensitivity and repeatability of MALDI-TOF-MSI, the CHCA matrix was selected. The matrix was used for MALDI-TOF-MSI in positive mode to analyze the distribution of coumarins in primary root and lateral root of A. dahurica. RESULTS: In total, 37 coumarins were detected in primary root and 36 coumarins were detected in lateral root by MALDI-TOF-MSI. The results showed that the coumarin content in primary root was higher than that in lateral root. Coumarins in primary root of A. dahurica were concentrated in the periderm, cortex, and phloem, whereas coumarins in lateral roots were concentrated in the phloem. CONCLUSION: The coumarins in primary root and lateral root of A. dahurica were directly analyzed without extraction and isolation, and the spatial distribution of coumarins was comprehensively visualized for the first time by MALDI-TOF-MSI, which provided a basis for distinguishing primary root and lateral root.

Mass spectrometry analysis of saponins.

Saponins are amphiphilic molecules of pharmaceutical interest and most of their biological activities (i.e., cytotoxic, hemolytic, fungicide, etc.) are associated to their membranolytic properties. These molecules are secondary metabolites present in numerous plants and in some marine animals, such as sea cucumbers and starfishes. Structurally, all saponins correspond to the combination of a hydrophilic glycan, consisting of sugar chain(s), linked to a hydrophobic triterpenoidic or steroidic aglycone, named the sapogenin. Saponins present a high structural diversity and their structural characterization remains extremely challenging. Ideally, saponin structures are best established using nuclear magnetic resonance experiments conducted on isolated molecules. However, the extreme structural diversity of saponins makes them challenging from a structural analysis point of view since, most of the time, saponin extracts consist in a huge number of congeners presenting only subtle structural differences. In the present review, we wish to offer an overview of the literature related to the development of mass spectrometry for the study of saponins. This review will demonstrate that most of the past and current mass spectrometry methods, including electron, electrospray and matrix-assisted laser desorption/ionization ionizations, gas/liquid chromatography coupled to (tandem) mass spectrometry, collision-induced dissociation including MS3 experiments, multiple reaction monitoring based quantification, ion mobility experiments, and so forth, have been used for saponin investigations with great success on enriched extracts but also directly on tissues using imaging methods.

The Use of Polydialkylsiloxanes/Triflic Acid as Derivatization Agents in the Analysis of Sulfur-Containing Aromatics by "Soft"-Ionization Mass Spectrometry.

Polycyclic aromatic sulfur-containing compounds are widely distributed in oil, especially in its low-volatile and heavy fractions (resins, asphaltenes), and this dictates the need for their determination when reliable methods for sulfur removing, cleaning and processing oil are developed. In these cases, "soft" ionization mass spectrometry methods, based on electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), are particularly effective. However, aromatic sulfur-containing compounds have low polarity and cannot be readily ionized by these methods. To overcome the problem, their preliminary conversion into sulfonium salts by the action of alkyl iodides and a silver-containing agent is widely used. In the process of developing more economical derivatization methods, we found a rather unexpected possibility of implementing S-alkylation of organic sulfides with commercial polydialkylsiloxanes (alkyl = CH3 or C2H5) in the presence of triflic acid (CF3SO3H) as a superacid co-alkylating agent. For homologous dibenzothiophenes as a typical model representative of petroleum sulfur-containing aromatic compounds, ESI and MALDI mass spectra exhibited the signals of corresponding S-alkylsulfonium salts with a high signal-to-noise ratio. A rational mechanism for the described chemical transformation is proposed, including the indispensable activation by triflic acid and the cleavage of the Si-C bond. Specific collision-induced dissociation of corresponding S-alkylated sulfonium cations is considered. The applicability of the derivatization approach to the analysis of petroleum products by high-resolution mass spectrometry is demonstrated.

Quantitative analysis capability of DIUTHAME mass spectrometry verified by acetylcholinesterase enzyme-catalyzed reaction assays.

Matrix-assisted laser desorption/ionization (MALDI) is advantageous for mass spectrometry applications where throughput is important. However, quantitative analysis is essentially problematic for MALDI-MS whose results depend on the intrinsically stochastic microcrystalline state of the matrix. High-throughput screening (HTS) of drug candidates is a typical example that requires high throughput. The application of MALDI-MS to HTS, which is quantitative analysis, imposes restrictions on designing an experimental system. Surface-assisted laser desorption/ionization (SALDI) methods, which do not depend on matrix crystal formation, are expected to be applied to quantitative analyses such as HTS. A recently developed one type of SALDI methods, desorption ionization using through hole alumina membrane (DIUTHAME), possesses a distinct feature that the surface microstructure effective for ionization is formed by through holes. In this study, the quantitative analysis capability of DIUTHAME was verified by applying DIUTHAME to enzyme-catalyzed reaction measurements, which are also used for HTS. Quantitative DIUTHAME-MS was conducted on various conditions of acetylcholinesterase-catalyzed reaction solutions containing cow milk as a substitute of biological media. Even for the enzyme-catalyzed reaction solutions containing complex additives that make the quantitative analysis extremely difficult, DIUTHAME based on the through hole structure enables quantitative measurements of the analytes by applying the reaction solutions to the back side of the laser exposed surface. In comparison with those obtained by MALDI-MS, the results obtained by DIUTHAME-MS showed less variability of data and delivered a better linearity of the Lineweaver-Burk plots and a more reasonable value of the Michaelis constant. Accordingly, it was demonstrated that DIUTHAME-MS possesses the quantitative analysis capability much better than that of MALDI-MS.

Ti3C2(OH)x-assisted LDI-TOF-MS for the rapid analysis of natural small molecules.

The inhomogeneous distribution of co-crystallized analytes and the traditional organic matrices as well as the intensive background interference in the low molecular weight range hinder the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) in the analysis of small-molecular compounds. New two-dimensional material MXene (e.g., Ti3C2) exerts better hydrophilicity, homogeneity and repeatability, and higher laser desorption efficiency, as well as less background interference than traditional organic matrices and other nanomaterial matrices such as titanium oxide, graphene, and gold nanostructures. This study was aimed to design Ti3C2 matrix with abundant hydroxyls on its surface, enhance the stability of this hydroxyl-rich Ti3C2 (Ti3C2(OH)x), and evaluate the analytical performances of Ti3C2(OH)x-assisted laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF-MS) for small-molecular natural compounds in complex samples. The developed Ti3C2(OH)x showed the distinct advantages such as minimum background interference, high peak intensity (~105), high salt (0.6 M) and protein (0.5 mg/mL) tolerance, good repeatability (relative standard deviation<20%), and good stability after eight months of storage. Ti3C2(OH)x-assisted LDI-TOF-MS analysis could be used to rapidly identify Artemisia annua (a world-famous traditional Chinese medicine) and quantify the contents of the main chemical ingredients (oxymatrine (OXY) and matrine) of Compound Kushen Injection (CKI). Interestingly, the content of OXY in CKI could be accurately quantified by Ti3C2(OH)x-assisted LDI-TOF-MS, and there was a good linear relationship (R2 -0.9929), a low limit of detection (400 pg), and a low limit of quantification (600 pg) of OXY. Taken together, the rapid and accurate analysis of small-molecular natural compounds in complicated samples could be achieved by the Ti3C2(OH)x-assisted LDI-TOF-MS analysis.

Mass spectrometry imaging: new eyes on natural products for drug research and development.

Natural products (NPs) and their structural analogs represent a major source of novel drug development for disease prevention and treatment. The development of new drugs from NPs includes two crucial aspects. One is the discovery of NPs from medicinal plants/microorganisms, and the other is the evaluation of the NPs in vivo at various physiological and pathological states. The heterogeneous spatial distribution of NPs in medicinal plants/microorganisms or in vivo can provide valuable information for drug development. However, few molecular imaging technologies can detect thousands of compounds simultaneously on a label-free basis. Over the last two decades, mass spectrometry imaging (MSI) methods have progressively improved and diversified, thereby allowing for the development of various applications of NPs in plants/microorganisms and in vivo NP research. Because MSI allows for the spatial mapping of the production and distribution of numerous molecules in situ without labeling, it provides a visualization tool for NP research. Therefore, we have focused this mini-review on summarizing the applications of MSI technology in discovering NPs from medicinal plants and evaluating NPs in preclinical studies from the perspective of new drug research and development (R&D). Additionally, we briefly reviewed the factors that should be carefully considered to obtain the desired MSI results. Finally, the future development of MSI in new drug R&D is proposed.

Porous Graphitic Carbon-Based Imprint Mass Spectrometry Imaging with an Ambient Liquid Extraction Technique for Enhancing Coverage of Glycerolipids and Sphingolipids in Brain Tissue.

Localization of lipidomes and tracking their spatial changes by mass spectrometry imaging (MSI) is critical for the mechanism studies on living process, disease, and therapeutic treatment. However, due to the strong ion suppression in complex biotissue, the imaging coverage for lipids with low polarity or low abundances, such as glycerolipids and sphingolipids, is usually limited. To address this issue, we utilized a porous graphitic carbon (PGC) material to imprint brain tissue sections for selective enrichment of neutral lipids with polar phospholipids removed. Then, the tissue imprint was scanned for desorption by the ambient liquid extraction MSI system. It was found that on the PGC surface, hydrophobic interaction dominates in protic solvents, and polar interaction dominates in aprotic solvents. Accordingly, methanol was selected as the spray solvent for tissue imprinting, and 75% acetonitrile-methanol was selected as the desorption solvent for the ambient liquid extraction MSI system. The results showed that glycerides had high recoveries after the imprinting-desorption process (recovery ∼ 70%) with phospholipids eliminated (recovery < 7%). To increase the transferring efficiencies of lipids from tissue onto PGC, electrospray was used for solvent application during imprinting, and the signals of diglycerides (DGs) in the imprint MSI of brain tissue increased by 2-3 times as compared to those via air spray. Finally, the new imprint MSI approach was applied to the imaging of the rat cerebellum and was compared with direct tissue MSI. The results showed that with imprint MSI, the coverage of DGs, sphingomyelins (SMs), and ceramides was enhanced by 4-5-fold (32 vs 6, 4 vs 1, and 5 vs 0). The ion images showed that with imprint MSI, higher signal intensities and clearer spatial distribution of DGs and SMs were obtained without interference from phosphatidylcholine signals compared with tissue MSI. This new method provides a complementary approach for traditional MSI to address the issues in imaging poorly ionizable or low-abundance lipids.

Determination of 15N/14N of Ammonium, Nitrite, Nitrate, Hydroxylamine, and Hydrazine Using Colorimetric Reagents and Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS).

In the nitrogen (N) cycle, nitrogenous compounds are chemically and biologically converted to various aqueous and gaseous N species. The 15N-labeling approach is a powerful culture-dependent technique to obtain insights into the complex nitrogen transformation reactions that occur in cultures. In the 15N-labeling approach, the fates of supplemented 15N- and/or unlabeled gaseous and aqueous compounds are tracked by mass spectrometry (MS) analysis, whereas MS analysis of aqueous N species requires laborious sample preparation steps and is performed using isotope-ratio mass spectrometry, which requires an expensive mass spectrometer. We developed a simple and high-throughput MS method for determining the 15N atoms percent of NH4+, NO2-, NO3-, NH2OH, and N2H4, where liquid samples (<0.5 mL) were mixed with colorimetric reagents (naphthylethylenediamine for NO2-, indophenol for NH4+, and p-aminobenzaldehyde for N2H4), and the mass spectra of the formed N complex dyes were obtained by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) MS. NH2OH and NO3- were chemically converted to NO2- by iodine oxidation and copper/hydrazine reduction reaction, respectively, prior to the above colorimetric reaction. The intensity of the isotope peak (M + 1 or M + 2) increased when the N complex dye was formed by coupling with a 15N-labeled compound, and a linear relationship was found between the determined 15N/14N peak ratio and 15N atom% for the tested N species. The developed method was applied to bacterial cultures to examine their N-transformation reactions, enabling us to observe the occurrence of NO2- oxidation and NO3- reduction in a hypoxic Nitrobacter winogradskyi culture. IMPORTANCE15N/14N analysis for aqueous N species is a powerful tool for obtaining insights into the global N cycle, but the procedure is cumbersome and laborious. The combined use of colorimetric reagents and MALDI-TOF MS, designated color MALDI-TOF MS, enabled us to determine the 15N atom% of common aqueous N species without laborious sample preparation and chromatographic separation steps; for instance, the 15N atom% of NO2- can be determined from >1,000 liquid samples daily at <$1 (U.S.) per 384 samples for routine analysis. This convenient MS method is a powerful tool that will advance our ability to explore the N-transformation reactions that occur in various environments and biological samples.