Spatial metabolomics using mass-spectrometry imaging to decipher the impact of high red meat diet on the colon metabolome in rat.

Colorectal cancer (CRC) is the third most common cancer in the world with a higher prevalence in the developed countries, mainly caused by environmental and lifestyle factors such as diet, particularly red meat consumption. The metabolic impact of high red meat consumption on the epithelial part of the colon was investigated using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MSI), to specifically analyze the epithelial substructure. Ten colons from rats fed for 100 days high red or white meat diet were subjected to untargeted MSI analyses using two spatial resolutions (100 µm and 10 µm) to evaluate metabolite changes in the epithelial part and to visualize the distribution of metabolites of interest within the epithelium crypts. Our results suggest a specific effect of red meat diet on the colonic epithelium metabolism, as evidenced by an increase of purine catabolism products or depletion in glutathione pool, reinforcing the hypothesis of increased oxidative stress with red meat diet. This study also highlighted cholesterol sulfate as another up-regulated metabolite, interestingly localized at the top of the crypts. Altogether, this study demonstrates the feasibility and the added value of using MSI to decipher the effect of high red meat diet on the colonic epithelium.

Evaluation of dried-blood spots and a hematocrit-independent procedure in lysosomal diseases screening using multiplexed tandem mass spectrometry assays.

BACKGROUND: Dried blood spots (DBS) are widely used as a non-invasive sampling method, especially in newborn screening (NBS). Despite its numerous advantages, conventional DBS might be limited by the hematocrit effect when analyzing a punch, depending on its position in the blood spot. This effect could be avoided using hematocrit-independent sampling devices such as the hemaPEN®. This device collects blood through integrated microcapillaries, and a fixed blood volume is deposited on a pre-punched paper disc. NBS programs are increasingly poised to include lysosomal disorders, given the availability of treatments that improve clinical outcomes if detected early. In this study, the effect of hematocrit and punch position in the DBS on the assay of 6 lysosomal enzymes was evaluated on 3 mm discs pre-punched in hemaPEN® devices compared to 3 mm punches from the PerkinElmer 226 DBS. METHODS: The enzyme activities were measured by multiplexed tandem mass spectrometry coupled to ultra-high performance liquid chromatography. Three hematocrit levels (23%, 35%, and 50%) and punching positions (center, intermediary, and border) were tested. Three replicates have been performed for each condition. A multivariate approach has been used along with a univariate method to assess the effect of the experimental design on each enzyme activity. RESULTS: Hematocrit, punch position, and whole blood sampling method do not affect the assessment of enzyme activity using the NeoLSD® assay. CONCLUSION: The results obtained from conventional DBS and the volumetric device HemaPEN® are comparable. These results underline the reliability of DBS for this test.

Large-Scale Interlaboratory DI-FT-ICR MS Comparability Study Employing Various Systems.

Ultrahigh resolution mass spectrometry (UHR-MS) coupled with direct infusion (DI) electrospray ionization offers a fast solution for accurate untargeted profiling. Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers have been shown to produce a wealth of insights into complex chemical systems because they enable unambiguous molecular formula assignment even if the vast majority of signals is of unknown identity. Interlaboratory comparisons are required to apply this type of instrumentation in quality control (for food industry or pharmaceuticals), large-scale environmental studies, or clinical diagnostics. Extended comparisons employing different FT-ICR MS instruments with qualitative direct infusion analysis are scarce since the majority of detected compounds cannot be quantified. The extent to which observations can be reproduced by different laboratories remains unknown. We set up a preliminary study which encompassed a set of 17 laboratories around the globe, diverse in instrumental characteristics and applications, to analyze the same sets of extracts from commercially available standard human blood plasma and Standard Reference Material (SRM) for blood plasma (SRM1950), which were delivered at different dilutions or spiked with different concentrations of pesticides. The aim of this study was to assess the extent to which the outputs of differently tuned FT-ICR mass spectrometers, with different technical specifications, are comparable for setting the frames of a future DI-FT-ICR MS ring trial. We concluded that a cluster of five laboratories, with diverse instrumental characteristics, showed comparable and representative performance across all experiments, setting a reference to be used in a future ring trial on blood plasma.

Imaging Matrix-Assisted Laser Desorption/Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry of oxaliplatin derivatives in human tissue sections.

Mass Spectrometry Imaging is an effective technology that allows to determine the in-situ distribution of endogen and/or exogen small molecules. It is a rapidly emerging approach for visualizing drugs and their metabolites within biological tissues. Matrix-Assisted Laser Desorption Ionization (MALDI) Mass Spectrometry Imaging (MSI) coupled to high resolving power analyzer (e.g. TOF) was already investigated for metallodrug localization and metabolization studies, but was proved to suffer from a lack of sensitivity and resolution, leading to poor coverage and assignment. To counter these technological limitations, the use of ultra-high resolving power analyzer such as Fourier Transform Ion Cyclotron Resonance (FTICR) could be revealed as a technique of choice. The high field FTICR MS provides ultra-high resolving power and mass accuracy that allows exhaustive molecule coverage and non-ambiguous molecular formula assignments. Platinum derivatives, such as oxaliplatin, are widely used as therapeutic agents for cancer treatment. The assessment of their intake, distribution and metabolism within the organs is important to know the risks associated with their use. In this study, MALDI FTICR MSI analyses were performed to better understand the penetration and metabolization of platinum derivatives in ovaries of women treated by Hyperthermic Intraperitoneal Chemotherapy (HIPEC) for peritoneal metastasis of colorectal or appendicular origin. Twelve ovary sections, from six ovary samples in six women donors, before and after treatment, were analyzed with 120 µm spatial resolution. For the first time, the high resolving power (220,000 at m/z 457) and sub-ppm accuracy (<1 ppm) of the FTICR combined with an Isotopic Fine Structure study enabled to distinguish two Pt-isobaric species derived from oxaliplatin in biological tissues. One of these, which is unknown, was specifically localized at the contour of the ovary.

Integrative Metabolomics Reveals Deep Tissue and Systemic Metabolic Remodeling in Glioblastoma.

(1) Background: Glioblastoma is the most common malignant brain tumor in adults. Its etiology remains unknown in most cases. Glioblastoma pathogenesis consists of a progressive infiltration of the white matter by tumoral cells leading to progressive neurological deficit, epilepsy, and/or intracranial hypertension. The mean survival is between 15 to 17 months. Given this aggressive prognosis, there is an urgent need for a better understanding of the underlying mechanisms of glioblastoma to unveil new diagnostic strategies and therapeutic targets through a deeper understanding of its biology. (2) Methods: To systematically address this issue, we performed targeted and untargeted metabolomics-based investigations on both tissue and plasma samples from patients with glioblastoma. (3) Results: This study revealed 176 differentially expressed lipids and metabolites, 148 in plasma and 28 in tissue samples. Main biochemical classes include phospholipids, acylcarnitines, sphingomyelins, and triacylglycerols. Functional analyses revealed deep metabolic remodeling in glioblastoma lipids and energy substrates, which unveils the major role of lipids in tumor progression by modulating its own environment. (4) Conclusions: Overall, our study demonstrates in situ and systemic metabolic rewiring in glioblastoma that could shed light on its underlying biological plasticity and progression to inform diagnosis and/or therapeutic strategies.

Muscle metabolic remodelling patterns in Duchenne muscular dystrophy revealed by ultra-high-resolution mass spectrometry imaging.

Duchenne muscular dystrophy (DMD) is a common and severe X-linked myopathy, characterized by muscle degeneration due to altered or absent dystrophin. DMD has no effective cure, and the underlying molecular mechanisms remain incompletely understood. The aim of this study is to investigate the metabolic changes in DMD using mass spectrometry-based imaging. Nine human muscle biopsies from DMD patients and nine muscle biopsies from control individuals were subjected to untargeted MSI using matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry. Both univariate and pattern recognition techniques have been used for data analysis. This study revealed significant changes in 34 keys metabolites. Seven metabolites were decreased in the Duchenne biopsies compared to control biopsies including adenosine triphosphate, and glycerophosphocholine. The other 27 metabolites were increased in the Duchenne biopsies, including sphingomyelin, phosphatidylcholines, phosphatidic acids and phosphatidylserines. Most of these dysregulated metabolites are tightly related to energy and phospholipid metabolism. This study revealed a deep metabolic remodelling in phospholipids and energy metabolism in DMD. This systems-based approach enabled exploring the metabolism in DMD in an unprecedented holistic and unbiased manner with hypothesis-free strategies.

Optimization of ion trajectories in a dynamically harmonized Fourier-transform ion cyclotron resonance cell using a design of experiments strategy.

RATIONALE: With the recent introduction of the dynamically harmonized Fourier-transform ion cyclotron resonance (FT-ICR) cell, the complexity of tuning has expanded drastically, and fine-tuning of the direct current voltages is required to optimize the ion cloud movement. As this adjustment must typically be performed manually, more reliable computational methods would be useful. METHODS: Here we propose a computational method based on a design of experiments (DoE) strategy to overcome the limits of classical manual tuning. This DoE strategy was exemplarily applied on a 12 T FT-ICR instrument equipped with a dynamically harmonized ICR cell. The chemometric approach, based on a central composite face (CCF) design, was first applied to a reference material (sodium trifluoroacetate) allowing for the evaluation of the primary cell parameters. Eight factors related to shimming and gating were identified. The summed intensity of the signal corresponding to the even harmonics was defined as one quality criterion. RESULTS: The DoE response allowed for rapid and complete mapping of cell parameters resulting in an optimized parameter set. The new set of cell parameters was applied to the study of an ultra-complex sample: Tholins, an ultra-complex mixture that mimics the haze present on Titan, was chosen. We observed a substantial improvement in mass spectrometric performance. The sum of signals related to harmonics was decreased by a factor of three (from 4% for conventional tuning to 1.3%). Furthermore, the dynamic range was also increased, which in turn led to an increase in attributed peaks by 13%. CONCLUSIONS: This computational procedure based on an experimental design can be applied to any other mass spectrometric parameter optimization problem. This strategy will lead to a more transparent and data-driven method development.

Monitoring of phosphorylation using immobilized kinases by on-line enzyme bioreactors hyphenated with High-Resolution Mass Spectrometry.

Nucleosides analogues are the cornerstone of the treatment of several human diseases. They are especially at the forefront of antiviral therapy. Their therapeutic efficiency depends on their capacity to be converted to the active nucleoside triphosphate form through successive phosphorylation steps catalyzed by nucleoside/nucleotide kinases. In this context, it is mandatory to develop a rapid, reliable and sensitive enzyme activity test to evaluate their metabolic pathways. In this study, we report a proof of concept to directly monitor on-line nucleotide multiple phosphorylation. The methodology was developed by on-line enzyme bioreactors hyphenated with High-Resolution Mass Spectrometry detection. Human Thymidylate Kinase (hTMPK) and human Nucleoside Diphosphate Kinase (hNDPK) were covalently immobilized on functionalized silica beads, and packed into micro-bioreactors (40 µL). By continuous infusion of substrate into the bioreactors, the conversion of thymidine monophosphate (dTMP) into its di- (dTDP) and tri-phosphorylated (dTTP) forms was visualized by monitoring their Extracted Ion Chromatogram (EIC) of their [M - H]- ions. Both bioreactors were found to be robust and durable over 60 days (storage at 4 °C in ammonium acetate buffer), after 20 uses and more than 750 min of reaction, making them suitable for routine analysis. Each on-line conversion step was shown rapid (<5 min), efficient (conversion efficiency > 55%), precise and repeatable (CV < 3% for run-to-run analysis). The feasibility of the on-line multi-step conversion from dTMP to dTTP was also proved. In the context of selective antiviral therapy, this proof of concept was then applied to the monitoring of specificity of conversion of two synthesized Acyclic Nucleosides Phosphonates (ANPs), regarding human Thymidylate Kinase (hTMPK) and vaccina virus Thymidylate Kinase (vvTMPK).

A new optimization strategy for MALDI FTICR MS tissue analysis for untargeted metabolomics using experimental design and data modeling.

Ultra-high-resolution imaging mass spectrometry using matrix-assisted laser desorption ionization (MALDI) MS coupled to a Fourier transform ion cyclotron resonance (FTICR) mass analyzer is a powerful technique for the visualization of small molecule distribution within biological tissues. The FTICR MS provides ultra-high resolving power and mass accuracy that allows large molecular coverage and molecular formula assignments, both essential for untargeted metabolomics analysis. These performances require fine optimizations of the MALDI FTICR parameters. In this context, this study proposes a new strategy, using experimental design, for the optimization of ion transmission voltages and MALDI parameters, for tissue untargeted metabolomics analysis, in both positive and negative ionization modes. These experiments were conducted by assessing the effects of nine factors for ion transmission voltages and four factors for MALDI on the number of peaks, the weighted resolution, and the mean error within m/z 150-1000 mass range. For this purpose, fractional factorial designs were used with multiple linear regression (MLR) to evaluate factor effects and to optimize parameter values. The optimized values of ion transmission voltages (RF amplitude TOF, RF amplitude octopole, frequency transfer optic, RF frequency octopole, deflector plate, funnel 1, skimmer, funnel RF amplitude, time-of-flight, capillary exit), MALDI parameters (laser fluence, number of laser shots), and detection parameters (data size, number of scans) led to an increase of 32% and 18% of the number of peaks, an increase of 8% and 39% of the resolution, and a decrease of 56% and 34% of the mean error in positive and negative ionization modes, respectively. Graphical abstract.

Monitoring of successive phosphorylations of thymidine using free and immobilized human nucleoside/nucleotide kinases by Flow Injection Analysis with High-Resolution Mass Spectrometry.

Nucleosides and their analogues play a crucial role in the treatment of several diseases including cancers and viral infections. Their therapeutic efficiency depends on their capacity to be converted to the active nucleoside triphosphates form through successive phosphorylation steps catalyzed by nucleoside/nucleotide kinases. It is thus mandatory to develop an easy, rapid, reliable and sensitive enzyme activity tests. In this study, we monitored the three-step phosphorylation of thymidine to thymidine triphosphate respectively by (1) human thymidine kinase 1 (hTK1), (2) human thymidylate kinase (hTMPK) and (3) human nucleoside diphosphate kinase (hNDPK). Free and immobilized kinase activities were characterized by using the Michaelis-Menten kinetic model. Flow Injection Analysis (FIA) with High-Resolution Mass Spectrometry (HRMS) was used as well as capillary electrophoresis (CE) with UV detection. The three-step cascade phosphorylation of thymidine was also monitored. FIA-HRMS allows a sensitive and rapid evaluation of the phosphorylation process. This study proposes simple, rapid, efficient and sensitive methods for enzyme kinetic studies and successive phosphorylation monitoring with immobilized enzymes.

TLC-UV hyphenated with MALDI-TOFMS for the screening of invertase substrates in plant extracts.

In this study, thin-layer chromatography (TLC) hyphenated with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was developed for the screening of invertase substrates in complex matrices. BfrA, a specific ß-D-fructofuranosidase from Leishmania major, was chosen as a model enzyme to screen biological activity in plant extracts due to its capacity to hydrolyze specific carbohydrates. TLC was considered to be a reliable technique for screening substrates (bioactive molecules) in plant extracts due to its quantitative capabilities whereas MALDI-TOFMS was particularly useful for rapid identification. The first part of this approach consisted of a differential analysis by TLC-densitometry to highlight band under- and over-expressions in plant extract between blank and enzymatic reaction. Zones of interest were then immediately analyzed by TLC-MALDI-TOFMS coupling to identify bioactive molecules. Development of the method presented various challenges: the separation and analysis of isomers (such as glucose and fructose), the high matrix effect (demonstrated by the analysis of products with invertase enzyme naturally present in plant extract), and the analysis of polar molecules with low molecular mass (sugars). Thanks to the separative technique, the specificity of detection, and the high precision of the characterization, this method was shown to be feasible for the analysis of bioactive molecules in complex mixtures containing interfering compounds (e.g. proteins, salts). Overall, this study demonstrates that Thin-layer chromatography hyphenated with Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is a simple, rapid, precise and efficient method for the analysis of suitable substrates in raw samples.

Validation of a thin-layer chromatography/densitometry method for the characterization of invertase activity.

This paper presents a kinetic study of invertase, a specific fructofuranosidase cloned from the Leishmania major genome. The kinetic parameters of the ß-d-fructofuranosidase from Leishmania major (BfrA) were determined using Thin-Layer Chromatography (TLC) and UV-densitometry (TLC@UV) specifically developed for the separation and detection of three carbohydrates namely sucrose, glucose and fructose. Separation was performed on TLC silica gel 60 F254 plates impregnated with sodium bisulphate and citrate and heated prior to development. This fast and easy separation was performed with two successive developments using ACN/H2O 80/20 (v/v) as mobile phase. Sensitive and repeatable derivatization of sugars was achieved by dipping the plates in a solution of 4-aminobenzoic acid. Quantification was performed by UV-detection. The method was validated according to ICH guidelines Q2(R1) in terms of specificity, limits of detection and quantification, precision and robustness (with n=3 replicates and CV ≤10%). The characterization of BfrA reaction kinetic was performed by monitoring the accumulation of either glucose or fructose detected by TLC@UV. Hydrolysis of sucrose was described by the Michaelis-Menten kinetic parameters (KM; Vmax) respectively equal to 63.09±7.590mM; 0.037±0.00094mM/min using glucose production and 83.01±14.39mM; 0.031±0.0021mM/min monitoring fructose. Hydrolyses of three alternative substrates, raffinose, stachyose and inulin, were also compared and the regiospecificity of the reaction was characterized. This TLC@UV method is shown to be suitable for the refined kinetic analysis of different reactions related to the hydrolysis of sugars.