[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.

MALDI-TOF Mass Spectrometry-Based Assay for Measuring Covalent Target Engagement of KRAS G12C Inhibitors.

MALDI-TOF mass spectrometry enables high-throughput screening of covalent fragment libraries and SAR compound progressions of selective KRAS G12C inhibitors. Using the MALDI-TOF platform instead of the more traditional ESI-MS TOF/orbitrap instrumentation can radically shorten sample acquisition time, allowing up to 384 samples to be screened in 30 min. The typical throughput for a covalent library screen is 1152 samples per 8 h, including processing, calculation, and reporting steps. The throughput can be doubled without any significant assay modification.

Imaging and quantification of neuropeptides in mouse pituitary tissue by atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry.

RATIONALE: Atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) mass spectrometry has enabled the untargeted analysis and imaging of neuropeptides and proteins in biological tissues under ambient conditions. Sensitivity in AP-MALDI can be improved by using sample-specific preparation methods. METHODS: A comprehensive and detailed optimization strategy including instrument parameters, matrix spraying and sample tissue washing pretreatment was implemented to enhance the sensitivity and coverage of neuropeptides in mouse pituitary tissues by commercial AP-MALDI mass spectrometry imaging (MSI). RESULTS: The sensitivity of a commercial AP-MALDI system for endogenous neuropeptides in mouse pituitary was enhanced by up to 15.2-fold by shortening the transmission gap from the sample plate to the inlet, attaching copper adhesive tape to an indium tin oxide-coated glass slide, optimizing the matrix spray solvent and using sample tissue washing pretreatment. Following careful optimization, the distributions of nine endogenous neuropeptides were successfully visualized in the pituitary. Furthermore, the quantitative capability of AP-MALDI for neuropeptides was evaluated and the concentrations of neuropeptides oxytocin and vasopressin in the pituitary posterior lobe were increased approximately twofold under hypertonic saline stress. CONCLUSION: Mouse pituitary neuropeptides have emerged as important signaling molecules due to their role in stress response. This work indicates the potential of modified AP-MALDI as a promising AP MSI method for in situ visualization and quantification of neuropeptides in complex biological tissues.

Absorption and translocation of selected pharmaceuticals in Pistia stratiotes: Spatial distribution analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Phytoremediation can eliminate pharmaceuticals from aquatic environments through absorption; however, understanding of absorption and transport processes in plants remains limited. In this study, a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-MSI) method was developed to explore the absorption and translocation mechanisms of seven common pharmaceuticals in Pistia stratiotes. Results showed that 2,3-dicyanohydroquinone, an infrequently used matrix, exhibited outstanding performance in MALDI-MSI analysis, producing the highest signal intensity for four of the seven pharmaceuticals. Region of Interest (ROI) analysis revealed that charge speciation of pharmaceuticals significantly influenced their ability to enter vascular bundle. Neutral and positively charged pharmaceuticals easily entered vascular bundle, while negatively charged pharmaceuticals faced difficulty. ROI results for neutral and negatively charged pharmaceuticals exhibited positive correlation with their transfer factor values, indicating that their translocation ability from root to shoot was related to their capacity to enter vascular bundle. However, no correlation was observed for positively charged pharmaceuticals, suggesting that these compounds, upon entering vascular bundle, encountered difficulties in upward translocation through the xylem. This study introduces an innovative approach and offers novel insights into the retention and migration of pharmaceuticals in plant tissues, aiming to enhance the understanding of pharmaceutical accumulation in plants. ENVIRONMENTAL IMPLICATION: Pharmaceuticals in aquatic environment can inflict detrimental effects on both human health and ecosystem. Phytoremediation can remove pharmaceuticals from aquatic environments through absorption. However, our understanding of absorption and transportation of pharmaceuticals in plants remains limited. This study developed a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-MSI) method for pharmaceuticals in plant roots, and to explore the absorption and translocation mechanisms of pharmaceuticals. The study offers direct evidence of differences in accumulation behavior of pharmaceuticals in plants, providing valuable insights for targeted and effective strategies in using plants for remediating the aquatic ecosystem from pharmaceuticals.

Multiple ion isolation and accumulation events for selective chemical noise reduction and dynamic range enhancement in MALDI imaging mass spectrometry.

Abundant chemical noise in MALDI imaging mass spectrometry experiments can impede the detection of less abundant compounds of interest. This chemical noise commonly originates from the MALDI matrix as well as other endogenous compounds present in high concentrations and/or with high ionization efficiencies. MALDI imaging mass spectrometry of biological tissues measures numerous biomolecular compounds that exist in a wide range of concentrations in vivo. When ion trapping instruments are used, highly abundant ions can dominate the charge capacity and lead to space charge effects that hinder the dynamic range and detection of lowly abundant compounds of interest. Gas-phase fractionation has been previously utilized in mass spectrometry to isolate and enrich target analytes. Herein, we have characterized the use of multiple continuous accumulations of selected ions (Multi CASI) to reduce the abundance of chemical noise and diminish the effects of space charge in MALDI imaging mass spectrometry experiments. Multi CASI utilizes the mass-resolving capability of a quadrupole mass filter to perform multiple sequential ion isolation events prior to a single mass analysis of the combined ion population. Multi CASI was used to improve metabolite and lipid detection in the MALDI imaging mass spectrometry analysis of rat brain tissue.

First reported human case of isolation of Vagococcus fluvialis from the urine of a former zoo clerk in Japan: a case report.

BACKGROUND: Vagococcal infections are extremely rare in humans. There are limited studies on the optimal methods for identification, antimicrobial susceptibility testing, and clinical manifestations of vagococcal infections. Herein, we report a patient with a urinary tract infection who had Vagococcus fluvialis in the urine. CASE PRESENTATION: An 84-year-old man presented to our urology department with a fever that had persisted for several days. He previously worked as a zoo clerk. The patient underwent a left nephroureterectomy for ureteral cancer 5 years ago, and total cystectomy and right cutaneous ureterostomy for muscle-invasive bladder cancer 1 year prior. He was empirically treated with 500 mg of levofloxacin intravenously every 24 h for the urinary tract infection. V. fluvialis was detected in his urine samples and Pseudomonas aeruginosa was detected in his urine and blood samples. Two bacterial species were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. He was administered intravenous levofloxacin for approximately 1 week, followed by oral levofloxacin for another week, after which the infections were eradicated. CONCLUSIONS: To the best of our knowledge, this is the first report of V. fluvialis detected in human urine in Japan. Vagococcus spp. is commonly isolated from fish or animals, and based on the patient's work history, it is possible that the patient was a carrier because of transmission from animals.

Imaging Mass Spectrometry of Isotopically Resolved Intact Proteins on a Trapped Ion-Mobility Quadrupole Time-of-Flight Mass Spectrometer.

In this work, we demonstrate rapid, high spatial, and high spectral resolution imaging of intact proteins by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) on a hybrid quadrupole-reflectron time-of-flight (qTOF) mass spectrometer equipped with trapped ion mobility spectrometry (TIMS). Historically, untargeted MALDI IMS of proteins has been performed on TOF mass spectrometers. While advances in TOF instrumentation have enabled rapid, high spatial resolution IMS of intact proteins, TOF mass spectrometers generate relatively low-resolution mass spectra with limited mass accuracy. Conversely, the implementation of MALDI sources on high-resolving power Fourier transform (FT) mass spectrometers has allowed IMS experiments to be conducted with high spectral resolution with the caveat of increasingly long data acquisition times. As illustrated here, qTOF mass spectrometers enable protein imaging with the combined advantages of TOF and FT mass spectrometers. Protein isotope distributions were resolved for both a protein standard mixture and proteins detected from a whole-body mouse pup tissue section. Rapid (∼10 pixels/s) 10 µm lateral spatial resolution IMS was performed on a rat brain tissue section while maintaining isotopic spectral resolution. Lastly, proof-of-concept MALDI-TIMS data was acquired from a protein mixture to demonstrate the ability to differentiate charge states by ion mobility. These experiments highlight the advantages of qTOF and timsTOF platforms for resolving and interpreting complex protein spectra generated from tissue by IMS.

Molecularly Imprinted Heterostructure-Assisted Laser Desorption Ionization Mass Spectrometry Analysis and Imaging of Quinolones.

Quinolone residues resulting from body metabolism and waste discharge pose a significant threat to the ecological environment and to human health. Therefore, it is essential to monitor quinolone residues in the environment. Herein, an efficient and sensitive matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) method was devised by using a novel molecularly imprinted heterojunction (MIP-TNs@GCNs) as the matrix. Molecularly imprinted titanium dioxide nanosheets (MIP-TNs) and graphene-like carbon nitrides (GCNs) were associated at the heterojunction interface, allowing for the specific, rapid, and high-throughput ionization of quinolones. The mechanism of MIP-TNs@GCNs was clarified using their adsorption properties and laser desorption/ionization capability. The prepared oxygen-vacancy-rich MIP-TNs@GCNs heterojunction exhibited higher light absorption and ionization efficiencies than TNs and GCNs. The good linearity (in the quinolone concentration range of 0.5-50 pg/µL, R2 > 0.99), low limit of detection (0.1 pg/µL), good reproducibility (n = 8, relative standard deviation [RSD] < 15%), and high salt and protein resistance for quinolones in groundwater samples were achieved using the established MIP-TNs@GCNs-MALDI/MS method. Moreover, the spatial distributions of endogenous compounds (e.g., amino acids, organic acids, and flavonoids) and xenobiotic quinolones from Rhizoma Phragmitis and Rhizoma Nelumbinis were visualized using the MIP-TNs@GCNs film as the MALDI/MS imaging matrix. Because of its superior advantages, the MIP-TNs@GCNs-MALDI/MS method is promising for the analysis and imaging of quinolones and small molecules.

MALDI-MSI-LC-MS/MS Workflow for Single-Section Single Step Combined Proteomics and Quantitative Lipidomics.

We introduce a novel approach for comprehensive molecular profiling in biological samples. Our single-section methodology combines quantitative mass spectrometry imaging (Q-MSI) and a single step extraction protocol enabling lipidomic and proteomic liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis on the same tissue area. The integration of spatially correlated lipidomic and proteomic data on a single tissue section allows for a comprehensive interpretation of the molecular landscape. Comparing Q-MSI and Q-LC-MS/MS quantification results sheds new light on the effect of MSI and related sample preparation. Performing MSI before Q-LC-MS on the same tissue section led to fewer protein identifications and a lower correlation between lipid quantification results. Also, the critical role and influence of internal standards in Q-MSI for accurate quantification is highlighted. Testing various slide types and the evaluation of different workflows for single-section spatial multiomics analysis emphasized the need for critical evaluation of Q-MSI data. These findings highlight the necessity for robust quantification methods comparable to current gold-standard LC-MS/MS techniques. The spatial information from MSI allowed region-specific insights within heterogeneous tissues, as demonstrated for glioblastoma multiforme. Additionally, our workflow demonstrated the efficiency of a single step extraction for lipidomic and proteomic analyses on the same tissue area, enabling the examination of significantly altered proteins and lipids within distinct regions of a single section. The integration of these insights into a lipid-protein interaction network expands the biological information attainable from a tissue section, highlighting the potential of this comprehensive approach for advancing spatial multiomics research.

Use of tryptic peptide MALDI mass spectrometry imaging to identify the spatial proteomic landscape of colorectal cancer liver metastases.

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide. CRC liver metastases (CRLM) are often resistant to conventional treatments, with high rates of recurrence. Therefore, it is crucial to identify biomarkers for CRLM patients that predict cancer progression. This study utilised matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to spatially map the CRLM tumour proteome. CRLM tissue microarrays (TMAs) of 84 patients were analysed using tryptic peptide MALDI-MSI to spatially monitor peptide abundances across CRLM tissues. Abundance of peptides was compared between tumour vs stroma, male vs female and across three groups of patients based on overall survival (0-3 years, 4-6 years, and 7+ years). Peptides were then characterised and matched using LC-MS/MS. A total of 471 potential peptides were identified by MALDI-MSI. Our results show that two unidentified m/z values (1589.876 and 1092.727) had significantly higher intensities in tumours compared to stroma. Ten m/z values were identified to have correlation with biological sex. Survival analysis identified three peptides (Histone H4, Haemoglobin subunit alpha, and Inosine-5'-monophosphate dehydrogenase 2) and two unidentified m/z values (1305.840 and 1661.060) that were significantly higher in patients with shorter survival (0-3 years relative to 4-6 years and 7+ years). This is the first study using MALDI-MSI, combined with LC-MS/MS, on a large cohort of CRLM patients to identify the spatial proteome in this malignancy. Further, we identify several protein candidates that may be suitable for drug targeting or for future prognostic biomarker development.

Peptide-Based Mass Spectrometry for the Investigation of Protein Complexes.

In the last two decades, biological mass spectrometry has become the gold standard for the identification of proteins in biological samples. The technological advancement of mass spectrometers and the development of methods for ionization, gas phase transfer, peptide fragmentation as well as for acquisition of high-resolution mass spectrometric data marked the success of the technique. This chapter introduces peptide-based mass spectrometry as a tool for the investigation of protein complexes. It provides an overview of the main steps for sample preparation starting from protein fractionation, reduction, alkylation and focus on the final step of protein digestion. The basic concepts of biological mass spectrometry as well as details about instrumental analysis and data acquisition are described. Finally, the most common methods for data analysis and sequence determination are summarized with an emphasis on its application to protein-protein complexes.

External quality control survey on identification of microorganisms using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry（ MALDI-TOF MS） is a bacterial typing tool that was approved as a medical device in 2011. However, external accuracy control examination of bacterial typing using mass spectrometry is still only performed on a small scale. In this study, E. faecium and S. maltophilia were selected and tested according to established procedures using Score Values at 228 institutions. The Score Values for MALDI Biotyper were 2.43±0.08 for E. faecium and 2.38±0.08 for S. maltophilia; and those for VITEK MS/PRIME were 99.9±0.0 for E. faecium and S. maltophilia. These results suggest that it is useful to evaluate external accuracy control with Score Values using the procedures we have developed.

Quantitative mass spectrometry imaging (qMSI): A tutorial.

Mass spectrometry imaging (MSI) is an analytical technique that enables the simultaneous detection of hundreds to thousands of chemical species while retaining their spatial information; usually, MSI is applied to biological tissues. Combining these elements can create ion images, which allows for the identification and localization of multiple chemical species within the sample. Being able to produce molecular images of biological tissues has already impacted the study of health and disease; however, the next logical step is being able to combine MSI with quantitative mass spectrometry methods to both quantify and determine the localization of disease progression or drug action. In this tutorial, we will detail the main factors to consider when designing a qMSI experiment and highlight the methods that have been developed to overcome these added complexities, specifically for those newer to the field of MSI.

Novel Small Molecule Matrix Screening for Simultaneous MALDI Mass Spectrometry Imaging of Multiple Lipids and Phytohormones.

Most of the traditional matrices cannot simultaneously image multiple lipids and phytohormones, so screening and discovery of novel matrices stand as essential approaches for broadening the application scope of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). In this work, 12 organic small molecule compounds were comprehensively screened and investigated as potential MALDI matrices for simultaneous imaging analysis of various lipids and phytohormones. In the positive ionization mode, p-nitroaniline, m-nitroaniline, and 2-aminoterephthalic acid displayed good performance for the highly sensitive detection of lysophosphatidylcholines (LPCs), phosphatidylcholines (PCs), and triacylglycerols (TGs). Furthermore, p-nitroaniline possessed excellent characteristics of strong ultraviolet absorption and homogeneous cocrystallization, making it a desirable matrix for MALDI-MSI analysis of eight plant hormones. Compared with conventional matrices (2,5-dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (CHCA), and 9-aminoacridine (9-AA), the use of p-nitroaniline resulted in higher ionization efficiency, superior sensitivity, and clearer imaging images in dual polarity mode. Our research offers valuable guidance and new ideas for future endeavors in matrix screening.

Functional Melanin Nanoparticles-Assisted Laser Desorption Ionization Mass Spectrometry for High-Sensitivity Detection of TBBPA and TBBPS Contaminations in Animal-Derived Foodstuffs.

Tetrabromobisphenol A (TBBPA) and tetrabromobisphenol S (TBBPS) have been widely used as additives in various products; however, their residues damage human health mainly via dietary ingestion. The current detection techniques remain challenging in directly and sensitively identifying TBBPA and TBBPS from food samples. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has great potential as an alternative tool for the analysis of low-mass environmental pollution. Herein, we successfully screened and optimized COOH-MNP-COOH as a novel MALDI matrix to enhance deprotonation for the analysis of TBBPA and TBBPS from animal-derived food samples in negative-ion mode. Notably, COOH-MNP-COOH was synthesized by a facile self-assembly strategy and characterized by TEM, FT-IR, UV-vis, and zeta potential analysis. Compared with conventional and control matrices, the COOH-MNP-COOH matrix exhibited excellent performance of TBBPA and TBBPS with high chemical stability, favorable reproducibility, remarkable salt and protein tolerance, and high sensitivity owing to abundant active groups, stronger UV-vis absorption at 355 nm, and better hydrophilicity and biocompatibility. TBBPA and TBBPS were detected with the assistance of an internal standard with limits of detection (LODs) of 300 and 200 pg/mL, respectively. Moreover, this method was applied to directly identify the residues of TBBPA and TBBPS in milk products, followed by basa catfish and meat. This research may provide a promising approach for the analysis of environmental pollutants in foodstuffs.

Mass spectrometry imaging protocol for spatial mapping of lipids, N-glycans and peptides in murine lung tissue.

RATIONALE: The application of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to murine lungs is challenging due to the spongy nature of the tissue. Lungs consist of interconnected air sacs (alveoli) lined by a single layer of flattened epithelial cells, which requires inflation to maintain its natural structure. Therefore, a protocol that is compatible with both lung instillation and high spatial resolution is essential to enable multi-omic studies on murine lung disease models using MALDI-MSI. METHODS AND RESULTS: To maintain the structural integrity of the tissue, murine lungs were inflated with 8% (w/v) gelatin for lipid MSI of fresh frozen tissues or 4% (v/v) paraformaldehyde neutral buffer for N-glycan and peptide MSI of FFPE tissues. Tissues were sectioned and prepared for enzymatic digestion and/or matrix deposition. Glycerol-free PNGase F was applied for N-glycan MSI, while Trypsin Gold was applied for peptide MSI using the iMatrixSpray and ImagePrep Station, respectively. For lipid, N-glycan and peptide MSI, α-cyano-4-hydroxycinnamic acid matrix was deposited using the iMatrixSpray. MS data were acquired with 20 µm spatial resolution using a timsTOF fleX MS instrument followed by MS fragmentation of lipids, N-glycans and peptides. For lipid MSI, trapped ion mobility spectrometry was used to separate isomeric/isobaric lipid species. SCiLS™ Lab was used to visualize all MSI data. For analyte identification, MetaboScape®, GlycoMod and Mascot were used to annotate MS fragmentation spectra of lipids, N-glycans and tryptic peptides, respectively. CONCLUSIONS: Our protocol provides instructions on sample preparation for high spatial resolution MALDI-MSI, MS/MS data acquisition and lipid, N-glycan and peptide annotation and identification from murine lungs. This protocol will allow non-biased analyses of diseased lungs from preclinical murine models and provide further insight into disease models.

Mass Spectrometry Imaging and Histology for the Analysis of Budding Intestinal Organoids.

Three-dimensional (3D) organoids have been at the forefront of regenerative medicine and cancer biology fields for the past decade. However, the fragile nature of organoids makes their spatial analysis challenging due to their budding structures and composition of single layer of cells. The standard sample preparation approaches can collapse the organoid morphology. Therefore, in this study, we evaluated several approaches to optimize a method compatible with both mass spectrometry imaging (MSI) and immunohistological techniques. Murine intestinal organoids were used to evaluate embedding in gelatin, carboxymethylcellulose (CMC)-gelatin-CMC-sucrose, or hydroxypropyl methylcellulose (HPMC) and polyvinylpyrrolidone (PVP) solutions. Organoids were assessed with and without aldehyde fixation and analyzed for lipid distributions by MSI coupled with hematoxylin and eosin (H&E) staining and immunofluorescence (IF) in consecutive sections from the same sample. While chemical fixation preserves morphology for better histological outcomes, it can lead to suppression of the matrix-assisted laser desorption/ionization (MALDI) lipid signal. By contrast, leaving organoid samples unfixed enhanced MALDI lipid signal. The method that performed best for both MALDI and histological analysis was embedding unfixed samples in HPMC and PVP. This approach allowed assessment of cell proliferation by Ki67 while also identifying putative phosphatidylethanolamine (PE(18:0/18:1)), which was confirmed further by tandem MS approaches. Overall, these protocols will be amenable to multiplexing imaging mass spectrometry analysis with several histological assessments and help advance our understanding of the biological processes that take place in district subsets of cells in budding organoid structures.

Visualization of PFOA accumulation and its effects on phospholipid in zebrafish liver by MALDI Imaging.

Exposure to poly- and perfluoroalkyl substances (PFASs) can result in bioaccumulation. Initial findings suggested that PFASs could accumulate in tissues rich in both phospholipids and proteins. However, our current understanding is limited to the average concentration of PFASs or phospholipid content across entire tissue matrices, leaving unresolved the spatial variations of lipid metabolism associated with PFOA in zebrafish tissue. To address gap, we developed a novel methodology for concurrent spatial profiling of perfluorooctanoic acid (PFOA) and individual phospholipids within zebrafish hepatic tissue sections, utilizing matrix-assisted laser desorption/ionization time of flight imaging mass spectrometry (MALDI-TOF-MSI). 5-diaminonapthalene (DAN) matrix and laser sensitivity of 50.0 were optimized for PFOA detection in MALDI-TOF-MSI analysis with high spatial resolution (25 µm). PFOA was observed to accumulate within zebrafish liver tissue. H&E staining results corroborating the damage inflicted by PFOA accumulation, consistent with MALDI MSI results. Significant up-regulation of 15 phospholipid species was observed in zebrafish groups exposed to PFOA, with these phospholipid demonstrating varied spatial distribution within the same tissue. Furthermore, co-localized imaging of distinct phospholipids and PFOA within identical tissue sections suggested there could be two distinct potential interactions between PFOA and phospholipids, which required further investigation. The MALDI-TOF-IMS provides a new tool to explore in situ spatial distributions and variations of the endogenous metabolites for the health risk assessment and ecotoxicology of emerging environmental pollutants.

Streamlining regular liquid chromatography with MALDI-TOF MS and NMR spectroscopy using automatic full-contact splitless spotting interface and flash-tap fractioning collection.

BACKGROUND: High-resolution matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and nuclear magnetic resonance (NMR) spectroscopy are powerful tools to identify unknown psychoactive substances. However, in complex matrices, trace levels of unknown substances usually require additional fractionation and concentration. Specialized liquid chromatography systems are necessary for both techniques. The small flow rate of nano LC, typically paired with MALDI-TOF MS, often results in prolonged fractionation times. Conversely, the larger flow rate of semi-preparative LC, used for NMR analysis, can be time-consuming and labor-intensive when concentrating samples. To address these issues, we developed an integrated automatic system that integrated to regular LC. RESULT: Automatic spot collector (ASC) and automatic fraction collector (AFC) were present in this study. The ASC utilized in-line matrix mixing, full-contact spotting and real time heating (50 °C), achieving great capacity of 5 µL droplet on MALDI plate, high recovery (76-116%) and rapid evaporation in 2 min. The analytes were concentrated 4-8 times, forming even crystallization, reaching the detection limit at the concentration of 50 µg L-1 for 12 psychoactive substances in urine. The AFC utilizes flexible tubing which flash-tapped the microtube's upper rim (3 mm depth) instead of reaching the bottom. This method prevents sample loss and minimizes the robotic arm's movement, providing a high fractionating speed at 6 s 12 psychoactive compounds were fractionated in a single round analysis (recovery: 81%-114%). Methamphetamine and nitrazepam obtained from drug-laced coffee samples were successful analyzed with photodiode array (PDA) after one AFC round and NMR after five rounds. SIGNIFICANCE: The ASC device employed real-time heating, in-line matrix mixing, and full-contact spotting to facilitate the samples spotting onto the MALDI target plate, thereby enhancing detection sensitivity in low-concentration and complex samples. The AFC device utilized the novel flash-tapping method to achieve rapid fractionation and high recovery rate. These devices were assembled using commercially available components, making them affordable (400 USD) for most laboratories while still meeting the required performance for advanced commercialized systems.

CSF N-Glycomics Using MALDI MS Techniques.

In this chapter, we will present the methodology currently applied in our laboratory for the structural elucidation of the cerebrospinal fluid (CSF) N-glycome. N-glycans are released from denatured carboxymethylated glycoproteins by digestion with peptide-N-glycosidase F (PNGase F) and purified using both C18 Sep-Pak® and porous graphitized carbon (PGC) HyperSep™ Hypercarb™ solid phase extraction (SPE) cartridges. The glycan pool is subsequently permethylated to increase mass spectrometry sensitivity. Molecular assignments are performed through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) analysis considering either the protein N-linked glycosylation pathway or MALDI TOF MS/MS data. Each stage has been optimized to obtain high-quality mass spectra in reflector mode with an optimal signal-to-noise ratio up to m/z 4800. This method has been successfully adopted to associate specific N-glycome profiles to the early and the advanced phases of Alzheimer's disease (AD).