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Context:Balanites aegyptiaca Del. (Zygophyllaceae) fruits are traditionally known for the treatment of hyperglycaemia. Several in vitro and in vivo studies proposed some mechanisms of action. However, clinical trials in human beings were never reported to date.Objectives: To investigate the antidiabetic efficacy of the 70% ethanol extract of the pericarps of B. aegyptiaca (BE) within a nutritional intervention in elderly people.Materials and methods: Ultra-performance electrospray ionization-mass spectroscopy (UPLC-ESI-MS/MS) analysis was used for metabolic profiling of BE which was incorporated in hard gelatine capsules (400 mg/day) and tested on 30 type 2 diabetes (T2D) Egyptian patients for 8 weeks. According to sex, age and body mass index participants were divided into two equivalent groups, placebo and treatment.Results: Thirteen compounds were identified in BE using UPLC-ESI-MS/MS analysis among which five steroidal saponins, seven phenolic compounds and a sterol glucoside. At the end of the 8-week treatment, the treated group showed 26.88% decrease in 2 h postprandial plasma glucose relative to 2.6% increase in the placebo group, while fasting plasma glucose was reduced to 10.3%. Treatment with BE capsules for 8 weeks produced significant reduction in the plasma triglyceride, total cholesterol and low-density lipoprotein cholesterol by 9.0, 12.76 and 21.35%, respectively, with 29.8% increase in the high-density lipoprotein cholesterol. Plasma alanine transaminase and aspartate transaminase were reduced by 42.6 and 43.3%, respectively.Discussion and conclusion: Administration of the BE capsules to T2D resulted in significant improvements in the glycaemic markers and the lipid profile, without adverse effects or hypoglycaemia.

Purpose Calcium folinate (leucovorin), which is converted in vivo into biologically active folate, enhances the potency of 5-fluorouracil (5-FU)-based chemotherapy in colorectal cancer. A common dosage of leucovorin in adjuvant and palliative settings is 60 mg/m 2 . The aim was to determine the levels of tetrahydrofolate (THF), 5,10-methylenetetrahydrofolate (methyleneTHF), and 5-methyltetrahydrofolate (methylTHF) in tumour and mucosa of colorectal cancer patients who received different dosages of leucovorin intravenously at time of surgery. Methods Eighty patients scheduled for colorectal resection with indication of colorectal cancer were randomised into four groups to receive leucovorin at 0, 60, 200, or 500 mg/m 2 , respectively. Blood samples were taken 10 and 30 min after leucovorin administration. Biopsy samples from tumour and mucosa were collected and snap-frozen at surgery. The levels of THF, methyleneTHF, and methylTHF in tumour and mucosa were assessed by liquid chromatography electrospray ionisation tandem mass spectrometry (LC–MS/MS) and the results were related to clinical diagnosis and therapeutic regimens. Results The folate levels in tissue revealed extensive inter-individual variability. The mean methyleneTHF value for the four treatment groups were 880, 1,769, 3,024 and 3,723 pmol/g ww . Only half of the patients who received 60 mg/m 2 leucovorin had higher levels of methyleneTHF in tumour than patients who received 0 mg/m 2 leucovorin. Rectal cancer patients had significantly lower levels of methyleneTHF compared with colon cancer patients. Conclusions There was a large inter-patient variability of tissue folate levels in colorectal cancer patients after supplementation with leucovorin at standardised dosage. High leucovorin doses were needed to exceed baseline methyleneTHF values, especially in rectal cancer patients. The results indicate that the standardised leucovorin dose may be insufficient to attain the full antitumour effect of 5-FU. Further studies are needed to establish whether higher dosage yields a better treatment response.

Electrospray ionization mass spectrometry (ESI-MS) is nowadays one of the cornerstones of biomolecular mass spectrometry and proteomics. Advances in sample preparation and mass analyzers have enabled researchers to extract much more information from biological samples than just the molecular weight. In particular, relevant for structural biology, noncovalent protein–protein and protein–ligand complexes can now also be analyzed by MS. For these types of analyses, assemblies need to be retained in their native quaternary state in the gas phase. This initial small niche of biomolecular mass spectrometry, nowadays often referred to as “native MS,” has come to maturation over the last two decades, with dozens of laboratories using it to study mostly protein assemblies, but also DNA and RNA-protein assemblies, with the goal to define structure–function relationships. In this perspective, we describe the origins of and (re)define the term native MS, portraying in detail what we meant by “native MS,” when the term was coined and also describing what it does (according to us) not entail. Additionally, we describe a few examples highlighting what native MS is, showing its successes to date while illustrating the wide scope this technology has in solving complex biological questions. Graphical Abstract ᅟ

Mass spectrometry imaging (MSI) enables label-free spatial mapping of hundreds of biomolecules in tissue sections. This capability provides valuable information on tissue heterogeneity that is difficult to obtain using population-averaged assays. Despite substantial developments in both instrumentation and methodology, MSI of tissue samples at single-cell resolution remains challenging. Herein, we describe a protocol for robust imaging of tissue sections with a high (better than 10-μm) spatial resolution using nanospray desorption electrospray ionization (nano-DESI) mass spectrometry, an ambient ionization technique that does not require sample pretreatment before analysis. In this protocol, mouse uterine tissue is used as a model system to illustrate both the workflow and data obtained in these experiments. We provide a detailed description of the nano-DESI MSI platform, fabrication of the nano-DESI and shear force probes, shear force microscopy experiments, spectral acquisition, and data processing. A properly trained researcher (e.g., technician, graduate student, or postdoc) can complete all the steps from probe fabrication to data acquisition and processing within a single day. We also describe a new strategy for acquiring both positive- and negative-mode imaging data in the same experiment. This is achieved by alternating between positive and negative data acquisition modes during consecutive line scans. Using our imaging approach, hundreds of high-quality ion images were obtained from a single uterine section. This protocol enables sensitive and quantitative imaging of lipids and metabolites in heterogeneous tissue sections with high spatial resolution, which is critical to understanding biochemical processes occurring in biological tissues.

Mass spectrometry is a central analytical technique for protein research and for the study of biomolecules in general. Driven by the need to identify, characterize, and quantify proteins at ever increasing sensitivity and in ever more complex samples, a wide range of new mass spectrometry-based analytical platforms and experimental strategies have emerged. Here we review recent advances in mass spectrometry instrumentation in the context of current and emerging research strategies in protein science.

Mass Spectrometry (MS) allows the analysis of proteins and peptides through a variety of methods, such as Electrospray Ionization-Mass Spectrometry (ESI-MS) or Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS). These methods allow identification of the mass of a protein or a peptide as intact molecules or the identification of a protein through peptide-mass fingerprinting generated upon enzymatic digestion. Tandem mass spectrometry (MS/MS) allows the fragmentation of proteins and peptides to determine the amino acid sequence of proteins (top-down and middle-down proteomics) and peptides (bottom-up proteomics). Furthermore, tandem mass spectrometry also allows the identification of post-translational modifications (PTMs) of proteins and peptides. Here, we discuss the application of MS/MS in biomedical research, indicating specific examples for the identification of proteins or peptides and their PTMs as relevant biomarkers for diagnostic and therapy.

Accurate identification of prostate cancer in frozen sections at the time of surgery can be challenging, limiting the surgeon’s ability to best determine resection margins during prostatectomy. We performed desorption electrospray ionization mass spectrometry imaging (DESI-MSI) on 54 banked human cancerous and normal prostate tissue specimens to investigate the spatial distribution of a wide variety of small metabolites, carbohydrates, and lipids. In contrast to several previous studies, our method included Krebs cycle intermediates (m/z <200), which we found to be highly informative in distinguishing cancer from benign tissue. Malignant prostate cells showed marked metabolic derangements compared with their benign counterparts. Using the “Least absolute shrinkage and selection operator” (Lasso), we analyzed all metabolites from the DESI-MS data and identified parsimonious sets of metabolic profiles for distinguishing between cancer and normal tissue. In an independent set of samples, we could use these models to classify prostate cancer from benign specimens with nearly 90% accuracy per patient. Based on previous work in prostate cancer showing that glucose levels are high while citrate is low, we found that measurement of the glucose/citrate ion signal ratio accurately predicted cancer when this ratio exceeds 1.0 and normal prostate when the ratio is less than 0.5. After brief tissue preparation, the glucose/citrate ratio can be recorded on a tissue sample in 1 min or less, which is in sharp contrast to the 20 min or more required by histopathological examination of frozen tissue specimens.

Modifications to an Orbitrap-based mass spectrometer enable analysis of large protein complexes in native-like states by mass spectrometry with very high sensitivity and mass resolution. The analysis of intact protein assemblies in native-like states by mass spectrometry offers a wealth of information on their biochemical and biophysical properties. Here we show that the Orbitrap mass analyzer can be used to measure protein assemblies of molecular weights approaching one megadalton with sensitivity down to the detection of single ions. Minor instrumental modifications enabled the measurement of various protein assemblies with outstanding mass-spectral resolution.

Tumors are comprised of a multitude of cell types spanning different microenvironments. Mass spectrometry imaging (MSI) has the potential to identify metabolic patterns within the tumor ecosystem and surrounding tissues, but conventional workflows have not yet fully integrated the breadth of experimental techniques in metabolomics. Here, we combine MSI, stable isotope labeling, and a spatial variant of Isotopologue Spectral Analysis to map distributions of metabolite abundances, nutrient contributions, and metabolic turnover fluxes across the brains of mice harboring GL261 glioma, a widely used model for glioblastoma. When integrated with MSI, the combination of ion mobility, desorption electrospray ionization, and matrix assisted laser desorption ionization reveals alterations in multiple anabolic pathways. De novo fatty acid synthesis flux is increased by approximately 3-fold in glioma relative to surrounding healthy tissue. Fatty acid elongation flux is elevated even higher at 8-fold relative to surrounding healthy tissue and highlights the importance of elongase activity in glioma. Isotopologue spectral analysis was originally designed to assess metabolic fluxes from bulk samples. Here, the authors adapted this approach to infer fluxes from discrete regions in tissue by using mass spectrometry imaging, showing increased fatty acid synthesis flux in brain tumors of mice.

This paper describes the validation of an LC-MS/MS-based method for the quantification of > 500 secondary microbial metabolites. Analytical performance parameters have been determined for seven food matrices using seven individual samples per matrix for spiking. Apparent recoveries ranged from 70 to 120% for 53–83% of all investigated analytes (depending on the matrix). This number increased to 84–94% if the recovery of extraction was considered. The comparison of the fraction of analytes for which the precision criterion of RSD ≤ 20% under repeatability conditions (for 7 replicates derived from different individual samples) and intermediate precision conditions (for 7 technical replicates from one sample), respectively, was met (85–97% vs. 93–94%) highlights the contribution of relative matrix effects to the method uncertainty. Statistical testing of apparent recoveries between pairs of matrices exhibited a significant difference for more than half of the analytes, while recoveries of the extraction showed a much better agreement. Apparent recoveries and matrix effects were found to be constant over 2–3 orders of magnitude of analyte concentrations in figs and maize, whereas the LOQs differed less than by a factor of 2 for 90% of the investigated compounds. Based on these findings, this paper discusses the applicability and practicability of current guidelines for multi-analyte method validation. Investigation of (apparent) recoveries near the LOQ seems to be insufficiently relevant to justify the enormous time-effort for manual inspection of the peaks of hundreds of analytes. Instead, more emphasis should be put on the investigation of relative matrix effects in the validation procedure.