Development and validation of an ultrafast method of quantification of rivaroxaban in human serum using laser diode thermal desorption coupled to triple quadrupole mass spectrometry.

RATIONALE: Rivaroxaban is an anticoagulant prescribed to patients who are at risk of medical conditions such as deep-vein thrombosis, pulmonary embolisms, and strokes caused by blood clots. The administration of this drug is monitored to adjust the dosage and evaluate patients' blood concentration. Rapid quantification of this drug in plasma could make it possible to ensure that the dose present in the blood of patients does not represent a danger for the medical intervention to be carried out. METHODS: Liquid chromatography-tandem mass spectrometry is usually employed to quantify rivaroxaban in blood, plasma, and serum. Here, an alternative method of analysis based on laser diode thermal desorption-triple quadrupole mass spectrometry (LDTD-QqQMS) was developed and comprehensively validated. This new method allows the quantification of rivaroxaban in less than 13 s from sample to sample. The extraction of rivaroxaban in human serum was done by a salting-out liquid-liquid extraction with acetonitrile and a saturated sodium chloride solution. RESULTS: The proposed method allows the quantification of rivaroxaban in less than 13 s from sample to sample. During validation, all criteria were respected. The accuracy was <15% of the nominal value, the precision was <15%CV, and the recovery was ≥89.9%. There were no observed carryover or matrix effects. Analysis of the extracted samples established the stability of dry (24 h) and wet samples (1 week) when samples cannot be analyzed immediately, a considerable advantage in a clinical setting. CONCLUSIONS: This method improves sample throughput by more than 1200% compared to liquid chromatography-tandem mass spectrometry methods of analysis of rivaroxaban and decreases analysis costs by reducing solvent consumption and instrument time.

Distribution of active ingredients and quality control of Forsythia suspensa with AP-MALDI mass spectrometry imaging.

The fruits of Forsythia suspensa (F. suspensa) have been used as a traditional Chinese medicine for 2000 years. Currently, the quality control of F. suspensa strictly follows the instructions of Chinese Pharmacopeia, which mainly controls the content of forsythoside A, phillyrin, and volatile oil. In this study, air pressure MALDI mass spectrometry imaging (AP-MALDI MSI) was used to evaluate the quality of F. suspensa fruits and the distribution of dozens of active ingredients. The variation of active ingredients was measured for more than 30 batches of samples, regarding harvest time, cultivated environment, shelf-life, and habitat. Fifty-three active ingredients could be detected in F. suspensa fruits with AP-MALDI MSI. Seven active ingredients were upregulated, four ingredients downregulated, and 15 ingredients did not change in ripe fruits. A sharp variation of active ingredients in late September was observed for the Caochuan fruits harvested in 2019, which is closely related to the appearance of the ginger color of the pericarp under the microscope observation. The microscope observation is a reliable way to classify ripe and green fruits instead of outlook. Just considering forsythoside A and phillyrin, it is found that wild fruits are better than cultivated fruits, but cultivated fruits have high contents of other ingredients. The shelf-life of F. suspensa fruits is proposed to be 3 years, considering the 26 ingredients investigated. It was found that Luoning wild fruits are better than those from Caochuan with a new evaluation method. Mass spectrometry imaging is an easy, objective, and effective method to evaluate the quality of F. suspensa fruits.

Investigation of Phosphatidylcholine by MALDI Imaging Mass Spectrometry in Normal and IVF Early-Stage Embryos.

The receptive phase of the uterus is marked by structural and functional maturation of the endometrium. During this limited time span, the blastocyst competency is superimposed on the receptive endometrium. It is a well-known fact that lipid signalling in early-stage pregnancy has a crucial role in successful embryogenesis. In our study, CD-1 mouse uteri after normal and in vitro fertilization (IVF) were investigated at 6.5, 8.5, and 10.5 days of pregnancy. Matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry and liquid chromatography coupled tandem mass spectrometry were used for identification of phosphatidylcholine (PC) lipid structures. In the embryonal tissues, PC 32:0 and PC 34:0 were increased, while in the antemesometrial (AM) decidua the two 20:4-containing PCs, PC 36:4 and PC 38:4 were increased. In transferred uterus samples, higher expressions of PC 34:0, PC 34:1, PC 34:2, PC 36:1, and PC 36:2 in mesometrial decidua were seen, whereas the two 20:4-containing PCs, PC 36:4 and PC 38:4 showed increased expression in the AM and lateral decidua. This paper shows a significant spatio-temporal change in lipid metabolism during IVF procedures for the first time.

Multi-modal mass spectrometry imaging of a single tissue section.

Mass spectrometry imaging (MSI) was developed to visualize spatial chemical information within tissues, thereby facilitating spatial multi-omic analysis. However, due to the limited spatial information provided by individual modal MSI, correlating various chemical data within tissues remains a significant challenge. In recent years, multimodal MSI has garnered considerable attention due to its ability to visualize the spatial distributions of multiple biomolecules within tissues. Among the strategies employed in this field, multimodal imaging on a single tissue section circumvents multiple issues introduced by integration of images of consecutive tissue sections. In this minireview, we provide an overview of multimodal MSI on a single tissue section, with a particular focus on the use of Matrix-Assisted Laser Desorption/Ionization-MSI for spatial multi-omic investigations that offer a comprehensive and in-depth elucidation of the biological state and activities, aiming to inspire the development of new approaches in this field.

Identification of Southeast Asian Anopheles mosquito species using MALDI-TOF mass spectrometry.

Malaria elimination in Southeast Asia remains a challenge, underscoring the importance of accurately identifying malaria mosquitoes to understand transmission dynamics and improve vector control. Traditional methods such as morphological identification require extensive training and cannot distinguish between sibling species, while molecular approaches are costly for extensive screening. Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid and cost-effective tool for Anopheles species identification, yet its current use is limited to few specialized laboratories. This study aimed to develop and validate an online reference database for MALDI-TOF MS identification of Southeast Asian Anopheles species. The database, constructed using the in-house data analysis pipeline MSI2 (Sorbonne University), comprised 2046 head mass spectra from 209 specimens collected at the Thailand-Myanmar border. Molecular identification via COI and ITS2 DNA barcodes enabled the identification of 20 sensu stricto species and 5 sibling species complexes. The high quality of the mass spectra was demonstrated by a MSI2 median score (min-max) of 61.62 (15.94-77.55) for correct answers, using the best result of four technical replicates of a test panel. Applying an identification threshold of 45, 93.9% (201/214) of the specimens were identified, with 98.5% (198/201) consistency with the molecular taxonomic assignment. In conclusion, MALDI-TOF MS holds promise for malaria mosquito identification and can be scaled up for entomological surveillance in Southeast Asia. The free online sharing of our database on the MSI2 platform (https://msi.happy-dev.fr/) represents an important step towards the broader use of MALDI-TOF MS in malaria vector surveillance.

Spatial distribution of metabolites in processing Ziziphi Spinosae Semen as revealed by matrix-assisted laser desorption/ionization mass spectrometry imaging.

Ziziphi Spinosae Semen (ZSS) is the first choice for the treatment of insomnia. This research aimed to reveal the spatial distribution of identifying quality markers of ZSS and to illustrate the metabolite quality characteristics of this herbal medicine. Here, we performed a matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) in situ to detect and image 33 metabolites in ZSS, including three saponins, six flavonoids, four alkaloids, eight fatty acids, and 12 amino acids. The MALDI images of the metabolites clearly showed the heterogeneous spatial distribution in different regions of ZSS tissues, such as the cotyledon, endosperm, and radicle. The distribution area of two saponins, six flavonoids, and three alkaloids increased significantly after the fried processing of ZSS. Based on the ion images, samples with different processing technologies were distinguished unambiguously by the pattern recognition method of orthogonal partial least squares discrimination analysis (OPLS-DA). Simultaneously, 23 major influencing components exerting higher ion intensities were identified as the potential quality markers of ZSS. Results obtained in the current research demonstrate that the processing of ZSS changes its content and distribution of the medicinal components. The analysis of MALDI-MSI provides a novel MS-based molecular imaging approach to investigate and monitor traditional medicinal plants.

Formic acid sandwich method is well-suited for filamentous fungi identification and improves turn around time using Zybio EXS2600 mass spectrometry.

OBJECTIVES: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is extensively employed for the identification of filamentous fungi on MALDI Biotyper (Bruker Daltonics) and Vitek MS (biomerieux), but the performance of fungi identification on new EXS2600 (Zybio) is still unknow. Our study aims to evaluate the new EXS2600 system's (Zybio) ability to rapidly identify filamentous fungi and determine its effect on turnaround time (TAT) in our laboratory. METHODS: We tested 117 filamentous fungi using two pretreatment methods: the formic acid sandwich (FA-sandwich) and a commercial mold extraction kit (MEK, Zybio). All isolates were confirmed via sequence analysis. Laboratory data were extracted from our laboratory information system over two 9-month periods: pre-EXS (April to December 2022) and post-EXS (April to December 2023), respectively. RESULTS: The total correct identification (at the species, genus, or complex/group level) rate of fungi was high, FA-sandwich (95.73%, 112/117), followed by MEK (94.02%, 110/117). Excluding 6 isolates not in the database, species-level identification accuracy was 92.79% (103/111) for FA-sandwich and 91.89% (102/111) for MEK; genus-level accuracy was 97.29% (108/111) and 96.39% (107/111), respectively. Both methods attained a 100% correct identification rate for Aspergillus, Lichtheimia, Rhizopus Mucor and Talaromyces species, and were able to differentiate between Fusarium verticillioides and Fusarium proliferatum within the Fusarium fujikuroi species complex. Notably, high confidence was observed in the species-level identification of uncommon fungi such as Trichothecium roseum and Geotrichum candidum. The TAT for all positive cultures decreased from pre EXS2600 to post (108.379 VS 102.438, P < 0.05), and the TAT for tissue decreased most (451.538 VS 222.304, P < 0.001). CONCLUSIONS: The FA-sandwich method is more efficient and accurate for identifying filamentous fungi with EXS2600 than the MEK. Our study firstly evaluated the performance of fungi identification on EXS2600 and showed it is suitable for clinical microbiology laboratories use.

[Determination of the derivatization reactivity between α/ß-dicarbonyl compounds and standard citrullinated peptides based on matrix-assisted laser desorption ionization-time-of-flight mass spectrometry].

Protein citrullination is an irreversible post-translational modification process regulated by peptidylarginine deiminases (PADs) in the presence of Ca2+. This process is closely related to the occurrence and development of autoimmune diseases, cancers, neurological disorders, cardiovascular and cerebrovascular diseases, and other major diseases. The analysis of protein citrullination by biomass spectrometry confronts great challenges owing to its low abundance, lack of affinity tags, small mass-to-charge ratio change, and susceptibility to isotopic and deamidation interferences. The methods commonly used to study the protein citrullination mainly involve the chemical derivatization of the urea group of the guanine side chain of the peptide to increase the mass-to-charge ratio difference of the citrullinated peptide. Affinity-enriched labels are then introduced to effectively improve the sensitivity and accuracy of protein citrullination by mass spectrometry. 2,3-Butanedione or phenylglyoxal compounds are often used as derivatization reagents to increase the mass-to-charge ratio difference of the citrullinated peptide, and the resulting derivatives have been observed to contain α-dicarbonyl structures. To date, however, no relevant studies on the reactivity of dicarbonyl compounds with citrullinated peptides have been reported. In this study, we determined whether six α-dicarbonyl and two ß-dicarbonyl compounds undergo derivatization reactions with standard citrullinated peptides using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Among the α-dicarbonyl compounds, 2,3-butanedione and glyoxal reacted efficiently with several standard citrullinated peptides, but yielded a series of by-products. Phenylglyoxal, methylglyoxal, 1,2-cyclohexanedione, and 1,10-phenanthroline-5,6-dione also derivated efficiently with standard citrullinated peptides, generating a single derivative. Thus, a new derivatization method that could yield a single derivative was identified. Among the ß-dicarbonyl compounds, 1,3-cyclohexanedione and 2,4-pentanedione successfully reacted with the standard citrullinated peptides, and generated a single derivative. However, their reaction efficiency was very low, indicating that the ß-dicarbonyl compounds are unsuitable for the chemical derivatization of citrullinated peptides. The above results indicate that the α-dicarbonyl structure is necessary for realizing the efficient and specific chemical derivatization of citrullinated peptides. Moreover, the side chains of the α-dicarbonyl structure determine the structure of the derivatives, derivatization efficiency, and generation (or otherwise) of by-products. Therefore, the specific enrichment and precise identification of citrullinated peptides can be achieved by synthesizing α-dicarbonyl structured compounds containing affinity tags. The proposed method enables the identification of citrullinated proteins and their modified sites by MS, thereby providing a better understanding of the distribution of citrullinated proteins in different tissues. The findings will be beneficial for studies on the mechanism of action of citrullinated proteins in a variety of diseases.

Highly Sensitive Spatial Glycomics at Near-Cellular Resolution by On-Slide Derivatization and Mass Spectrometry Imaging.

Glycans on proteins and lipids play important roles in maturation and cellular interactions, contributing to a variety of biological processes. Aberrant glycosylation has been associated with various human diseases including cancer; however, elucidating the distribution and heterogeneity of glycans in complex tissue samples remains a major challenge. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is routinely used to analyze the spatial distribution of a variety of molecules including N-glycans directly from tissue surfaces. Sialic acids are nine carbon acidic sugars that often exist as the terminal sugars of glycans and are inherently difficult to analyze using MALDI-MSI due to their instability prone to in- and postsource decay. Here, we report on a rapid and robust method for stabilizing sialic acid on N-glycans in FFPE tissue sections. The established method derivatizes and identifies the spatial distribution of α2,3- and α2,6-linked sialic acids through complete methylamidation using methylamine and PyAOP ((7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate). Our in situ approach increases the glycans detected and enhances the coverage of sialylated species. Using this streamlined, sensitive, and robust workflow, we rapidly characterize and spatially localize N-glycans in human tumor tissue sections. Additionally, we demonstrate this method's applicability in imaging mammalian cell suspensions directly on slides, achieving cellular resolution with minimal sample processing and cell numbers. This workflow reveals the cellular locations of distinct N-glycan species, shedding light on the biological and clinical significance of these biomolecules in human diseases.

Alternative protocol leading to rapid identification of Actinomycetes isolated from Algerian desertic soil by MALDI-TOF mass spectrometry.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is the first-line method for the rapid identification of most cultured microorganisms. As for Streptomyces strains, MALDI-TOF MS identification is complicated by the characteristic incrustation of colonies in agar and the strong cell wall of Actinomycetes cells requiring the use of alternative protein extraction protocols. In this study, we developed a specific protocol to overcome these difficulties for the MALDI-TOF MS identification of Actinomycetes made on solid medium. This protocol includes incubation of colony removed from agar plate with the beta-agarase enzyme, followed by a mechanical lysis and two washes by phosphate buffer and ethanol. Twenty-four Streptomyces and two Lentzea strains isolated from Algerian desertic soils were first identified by 16S rRNA sequencing as gold standard method, rpoB gene was used as a secondary gene target when 16S rRNA did not allow species identification. In parallel the isolates were identified by using the MALDI-TOF MS protocol as reported. After the expansion of the database with the inclusion of this MSPS, the strains were analyzed again in MALDI Biotyper, and all were identified. This work demonstrates that the rapid identification of Actinomycetes can be obtained without protein extraction step frequently used in MALDI-TOF mass spectrometry with this type of microorganisms.

Diagnosis of epilepsy by machine learning of high-performance plasma metabolic fingerprinting.

Epilepsy is a chronic neurological disorder that causes a major threat to public health and the burden of disease worldwide. High-performance diagnostic tools for epilepsy need to be developed to improve diagnostic accuracy and efficiency while still missing. Herein, we utilized nanoparticle-enhanced laser desorption/ionization mass spectrometry (NELDI MS) to acquire plasma metabolic fingerprints (PMFs) from epileptic and healthy individuals for timely and accurate screening of epilepsy. The NELDI MS enabled high detection speed (∼30 s per sample), high throughput (up to 384 samples per run), and favorable reproducibility (coefficients of variation <15 %), acquiring high-performed PMFs. We next constructed an epilepsy diagnostic model by machine learning of PMFs, achieving desirable diagnostic capability with the area under the curve (AUC) value of 0.941 for the validation set. Furthermore, four metabolites were identified as a diagnostic biomarker panel for epilepsy, with an AUC value of 0.812-0.860. Our approach provides a high-performed and high-throughput platform for epileptic diagnostics, promoting the development of metabolic diagnostic tools in precision medicine.

Gel-assisted mass spectrometry imaging enables sub-micrometer spatial lipidomics.

A technique capable of label-free detection, mass spectrometry imaging (MSI) is a powerful tool for spatial investigation of native biomolecules in intact specimens. However, MSI has often been precluded from single-cell applications due to the spatial resolution limit set forth by the physical and instrumental constraints of the method. By taking advantage of the reversible interaction between the analytes and a superabsorbent hydrogel, we have developed a sample preparation and imaging workflow named Gel-Assisted Mass Spectrometry Imaging (GAMSI) to overcome the spatial resolution limits of modern mass spectrometers. With GAMSI, we show that the spatial resolution of MALDI-MSI can be enhanced ~3-6-fold to the sub-micrometer level without changing the existing mass spectrometry hardware or analysis pipeline. This approach will vastly enhance the accessibility of MSI-based spatial analysis at the cellular scale.

Large-Scale Screening of Pharmaceutical Compounds to Explore the Application Space of On-Tissue MALDI and MALDI-2 Mass Spectrometry.

The successful application of matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) in pharmaceutical research is strongly dependent on the detection of the drug of interest at physiologically relevant concentrations. Here we explored how insufficient sensitivity due to low ionization efficiency and/or the interaction of the drug molecule with the local biochemical environment of the tissue can be mitigated for many compound classes using the recently introduced MALDI-MSI coupled with laser-induced postionization, known as MALDI-2-MSI. Leveraging a MALDI-MSI screen of about 1,200 medicines/drug-like compounds from a broad range of medicinal application areas, we demonstrate a significant improvement in drug detection and the degree of sensitivity uplift by using MALDI-2 versus traditional MALDI. Our evaluation was made under simulated imaging conditions using liver homogenate sections as substrate, onto which the compounds were spotted to mimic biological conditions to the first order. To enable an evaluable detection by both MALDI and MALDI-2 for the majority of employed compounds, we spotted 1 µL of a 10 mM solution using a spotting robot and performed our experiments with a Bruker timsTOF fleX MALDI-2 instrument in both positive and negative ion modes. Specifically, we demonstrate using a large cohort of drug-like compounds that ∼60% of the tested compounds showed a more than 10-fold increase in signal intensity and ∼16% showed a more than 100-fold increase upon use of MALDI-2 postionization. Such increases in sensitivity could help advance pharmaceutical MALDI-MSI applications toward the single-cell level.

Co-registration of MALDI-MSI and histology demonstrates gangliosides co-localize with amyloid beta plaques in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurological condition characterized by impaired cognitive function and behavioral alterations. While AD research historically centered around mis-folded proteins, advances in mass spectrometry techniques have triggered increased exploration of the AD lipidome with lipid dysregulation emerging as a critical player in AD pathogenesis. Gangliosides are a class of glycosphingolipids enriched within the central nervous system. Previous work has suggested a shift in a-series gangliosides from complex (GM1) to simple (GM2 and GM3) species may be related to the development of neurodegenerative disease. In addition, complex gangliosides with 20 carbon sphingosine chains have been shown to increase in the aging brain. In this study, we utilized matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) to interrogate the in situ relationship of a-series gangliosides with either 18 or 20 carbon sphingosine chains (d18:1 or d20:1, respectively) in the post-mortem human AD brain. Here, we expanded upon previous literature and demonstrated a significant decrease in the GM1 d20:1 to GM1 d18:1 ratio in regions of the dentate gyrus and entorhinal cortex in AD relative to control brain tissue. Then, we demonstrated that the MALDI-MSI profile of GM3 co-localizes with histologically confirmed amyloid beta (Aß) plaques and found a significant increase in both GM1 and GM3 in proximity to Aß plaques. Collectively, this study demonstrates a perturbation of the ganglioside profile in AD, and validates a pipeline for MALDI-MSI and classic histological staining in the same tissue sections. This demonstrates feasibility for integrating untargeted mass spectrometry imaging approaches into a digital pathology framework.

A Simple and Rapid Microscale Method for Isolating Bacterial Lipopolysaccharides.

Bacterial endotoxins (lipopolysaccharides (LPSs)) are important mediators of inflammatory processes induced by Gram-negative microorganisms. LPSs are the key inducers of septic shock due to a Gram-negative bacterial infection; thus, the structure and functions of LPSs are of specific interest. Often, highly purified bacterial endotoxins must be isolated from small amounts of biological material. Each of the currently available methods for LPS extraction has certain limitations. Herein, we describe a rapid and simple microscale method for extracting LPSs. The method consists of the following steps: ultrasonic destruction of the bacterial material, LPS extraction via heating, LPS purification with organic solvents, and treatment with proteinase K. LPSs that were extracted by using this method contained less than 2-3% protein and 1% total nucleic acid. We also demonstrated the structural integrity of the O-antigen and lipid A via the sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) methods, respectively. We demonstrated the ability of the extracted LPSs to induce typical secretion of cytokines and chemokines by primary macrophages. Overall, this method may be used to isolate purified LPSs with preserved structures of both the O-antigen and lipid A and unchanged functional activity from small amounts of bacterial biomass.

Role of Chalcogenides in Sensitive Therapeutic Drug Monitoring Using Laser Desorption and Ionization.

This study investigates the applicability of six transition metal dichalcogenides to efficient therapeutic drug monitoring of ten antiepileptic drugs using laser desorption/ionization-mass spectrometry. We found that molybdenum ditelluride and tungsten ditelluride are suitable for the sensitive quantification of therapeutic drugs. The contribution of tellurium to the enhanced efficiency of laser desorption ionization was validated through theoretical calculations utilizing an integrated model that incorporates transition-metal dichalcogenides and antiepileptic drugs. The results of our theoretical calculations suggest that the relatively low surface electron density for the tellurium-containing transition metal dichalcogenides induces stronger Coulombic interactions, which results in enhanced laser desorption and ionization efficiency. To demonstrate applicability, up to 120 patient samples were analyzed to determine drug concentrations, and the results were compared with those of immunoassay and liquid chromatography-tandem mass spectrometry. Agreements among these methods were statistically evaluated using the Passing-Bablok regression and Bland-Altman analysis. Furthermore, our method has been shown to be applicable to the simultaneous detection and multiplexed quantification of antiepileptic drugs.

Revealing Glycosylation Patterns in In Vitro-Produced Mucus Exposed to Pasteurized Mucus-Associated Intestinal Microbes by MALDI-TOF-MS and PGC-LC-MS/MS.

The human intestinal mucus layer protects against pathogenic microorganisms and harmful substances, whereas it also provides an important colonization niche for mutualistic microbes. The main functional components of mucus are heavily glycosylated proteins, called mucins. Mucins can be cleaved and utilized by intestinal microbes. The mechanisms between intestinal microbes and the regulation of mucin glycosylation are still poorly understood. In this study, in vitro mucus was produced by HT29-MTX-E12 cells under Semi-Wet interface with Mechanical Stimulation. Cells were exposed to pasteurized nonpathogenic bacteria Akkermansia muciniphila, Ruminococcus gnavus, and Bacteroides fragilis to evaluate influence on glycosylation patterns. Following an optimized protocol, O- and N-glycans were efficiently and reproducibly released, identified, and semiquantified using MALDI-TOF-MS and PGC-LC-MS/MS. Exposure of cells to bacteria demonstrated increased diversity of sialylated O-glycans and increased abundance of high mannose N-glycans in in vitro produced mucus. Furthermore, changes in glycan ratios were observed. It is speculated that bacterial components interact with the enzymatic processes in glycan production and that pasteurized bacteria influence glycosyltransferases or genes involved. These results highlight the influence of pasteurized bacteria on glycosylation patterns, stress the intrinsic relationship between glycosylation and microbiota, and show the potential of using in vitro produced mucus to study glycosylation behavior.

A Simple and Rapid Quantitative Assay for Gossypol via Reactive Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

The development of simple and rapid analytical tools for gossypol (GSP) is important to the food industry and medical field. Here, we report a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method for the detection of GSP by using a reactive matrix 4-hydrazinoquinazoline (4-HQ). The two aldehyde groups of GSP react with the 4-HQ and therefore improve the detection sensitivity and selectivity of GSP. Moreover, GSP forms homogeneous crystals with the 4-HQ matrix, allowing the quantification of the GSP by the proposed method. With the optimized experimental conditions, GSP could be detected at concentrations as low as 0.1 µM and quantified in a wide linear range (1-500 µM). After a brief extraction with an organic solvent, the GSP contents in cottonseeds and cottonseed kernels from different provinces of China were determined successfully. The spiked recovery of GSP in cottonseed/cottonseed kernel samples was obtained as 97.88-105.80%, showing the reliability of the assay for GSP determination in real samples.

Feasibility of MALDI-MSI-Based Proteomics Using Bouin-Fixed Pathology Samples: Untapping the Goldmine of Nephropathology Archives.

The application of innovative spatial proteomics techniques, such as those based upon matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) technology, has the potential to impact research in the field of nephropathology. Notwithstanding, the possibility to apply this technology in more routine diagnostic contexts remains limited by the alternative fixatives employed by this ultraspecialized diagnostic field, where most nephropathology laboratories worldwide use bouin-fixed paraffin-embedded (BFPE) samples. Here, the feasibility of performing MALDI-MSI on BFPE renal tissue is explored, evaluating variability within the trypsin-digested proteome as a result of different preanalytical conditions and comparing them with the more standardized formalin-fixed paraffin-embedded (FFPE) counterparts. A large proportion of the features (270, 68.9%) was detected in both BFPE and FFPE renal samples, demonstrating only limited variability in signal intensity (10.22-10.06%). Samples processed with either fixative were able to discriminate the principal parenchyma regions along with diverse renal substructures, such as glomeruli, tubules, and vessels. This was observed when performing an additional "stress test", showing comparable results in both BFPE and FFPE samples when the distribution of several amyloid fingerprint proteins was mapped. These results suggest the utility of BFPE tissue specimens in MSI-based nephropathology research, further widening their application in this field.

Quantitative Analysis of Blended Oils Based on Intensity Ratios of Marker Ions in MALDI-MS Spectra.

Determination of quantitative compositions of blended oils is an essential but challenging step for the quality control and safety assurance of blended oils. We herein report a method for the quantitative analysis of blended oils based on the intensity ratio of triacylglycerol marker ions, which could be obtained from the highly reproducible spectra acquired by using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to directly analyze blended oils in their oily states. We demonstrated that this method could provide good quantitative results to binary, ternary, and quaternary blended oils, with simultaneous quantitation of multiple compositions, and was applicable for quantitative analysis of commercial blended oil products. Moreover, the intensity ratio-based method could be used to rapidly measure the proportions of oil compositions in blended oils, only based on the spectra of the blended oils and related pure oils, making the method as a high-throughput approach to meet the sharply growing analytical demands of blended oils.