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.

Evaluation of the environmental contamination and exposure risk in medical/non-medical staff after oxaliplatin-based pressurized intraperitoneal aerosol chemotherapy.

Pressurized intraperitoneal aerosol chemotherapy (PIPAC) is a technique to directly deliver chemotherapeutic drugs in the abdomen for the treatment of peritoneal metastases. Pressurization improves the treatment efficacy but increases the risk of exposure for the medical/non-medical staff who can be exposed by dermal or ocular contact, or inhalation of aerosols containing the cytotoxic drugs. The aim of this study was to evaluate the risk of exposure for the medical/non-medical staff (nurses, surgeons, anaesthesiologists and cleaning personnel; n = 13) during PIPAC with oxaliplatin performed according to the protocol recommended in France. Blood samples were collected 1 h before and immediately after PIPAC, and urine samples 1 h before, and then 3 h and the morning after PIPAC. In the control, non-exposed group (n = 7), only one urine and blood sample were collected. Surface contamination in the operating room was assessed in water- and Surfanios-impregnated wipe samples. The total elemental platinum in each sample was quantified by inductively coupled plasma mass spectrometry, using a method adapted to quantify trace amounts (ng.L-1) in very low volumes (100 µl). No surface contamination was detected. Although 25% of urine samples in the exposed group contained platinum, no statistical difference was observed in urine and plasma samples collected before and after PIPAC and with the control group samples. These findings suggest that the French PIPAC protocol does not increase the risk of exposure to platinum in all staff categories involved. This protocol could be considered in future occupational policies and consensus statements. Trial registration: NCT04014426.

Study of oxaliplatin penetration into ovaries of patients treated with hyperthermic intraperitoneal chemotherapy (HIPEC) for peritoneal metastases of colorectal and appendiceal origin using mass spectrometry imaging.

OBJECTIVES: Platinum salts are commonly used in hyperthermic intraperitoneal chemotherapy (HIPEC) for digestive tract cancer treatment. During HIPEC with oxaliplatin for peritoneal metastases (PMs) treatment, the ovaries are directly exposed to the drug, questioning about ovarian resection and the potential impact of the drug on ovarian functionality, especially in young women of childbearing age. The goal of this work is to understand unwanted damages to the ovaries during HIPEC therapy by the determination of the concentration and distribution of platinum in ovaries in order to address its potential toxicity. METHODS: Mass spectrometry imaging techniques, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP MS), were used to study the penetration of oxaliplatin in ovaries after HIPEC treatment. RESULTS: MALDI-MS allowed the localization of an oxaliplatin-derivative (m/z 456.2) at the periphery of the ovaries. The quantitative LA-ICP MS maps confirmed the localization of elemental platinum as well as in the central part of ovaries from patients who received a previous platinum salt-based chemotherapy. CONCLUSIONS: LA-ICP MS images showed that platinum diffusion was extended in cases of previous systemic treatment, questioning about platinum derivatives gonado-toxicity when combining the two treatments.

Trace element distribution in marine microplastics using laser ablation-ICP-MS.

Due to the dramatic quantity of plastic debris released into our environment, one of the biggest challenges of the next decades is to trace and quantify microplastics (MPs) in our environments, especially to better evaluate their capacity to transport other contaminants such as trace metals. In this study, trace elements (Fe, Cu, Zn, As, Cd, Sn, Sb, Pb, and U) were analyzed in the microplastic subsurface (200 µm) using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Microplastics subjected to the marine environment were collected on beaches (Guadeloupe) exposed to the north Atlantic gyre. We established a strategy to discriminate sorbed contaminants from additives based on the metal concentration profiles in MP subsurface using qualitative and quantitative approaches. A spatiotemporal correlation of the sorption pattern was proposed to compare MPs in terms of relative exposure time and time-weighted average concentrations in the exposure media.

Cadmium distribution in mature durum wheat grains using dissection, laser ablation-ICP-MS and synchrotron techniques.

Understanding how essential and toxic elements are distributed in cereal grains is a key to improving the nutritional quality of cereal-based products. The main objective of this work was to characterize the distribution of Cd and of nutrients (notably Cu, Fe, Mn, P, S and Zn) in the durum wheat grain. Laser ablation inductively coupled mass spectrometry and synchrotron micro X-ray fluorescence were used for micro-scale mapping of Cd and nutrients. A dissection approach was used to quantitatively assess the distribution of Cd and nutrients among grain tissues. Micro X-ray absorption near-edge spectroscopy was used to identify the Cd chemical environment in the crease. Cadmium distribution was characterized by strong accumulation in the crease and by non-negligible dissemination in the endosperm. Inside the crease, Cd accumulated most in the pigment strand where it was mainly associated with sulfur ligands. High-resolution maps highlighted very specific accumulation areas of some nutrients in the germ, for instance Mo in the root cortex primordia and Cu in the scutellum. Cadmium loading into the grain appears to be highly restricted. In the grain, Cd co-localized with several nutrients, notably Mn and Zn, which challenges the idea of selectively removing Cd-enriched fractions by dedicated milling process.

The impact of fermentation on the distribution of cadmium in cacao beans.

A large fraction of the South-American cacao production is affected by new cadmium (Cd) regulations in cacao. This work was set up to characterize the distribution and speciation of Cd within the cacao fruit and to monitor potential Cd redistribution during cacao fermentation. In cacao fruits from four locations, Cd concentrations decreased with testa > nib ~ placenta ~ pod husk > mucilage. The distribution of Cd within cacao beans was successfully visualized using laser ablation inductively coupled plasma spectrometry (LA-ICP-MS) and confirmed higher Cd concentrations in the testa than in the nib. Speciation analysis by X-ray absorption spectroscopy (XANES) of unfermented cacao beans revealed that Cd was bound to O/N-ligands in both nib and testa. Fermentation induced an outward Cd migration from the nibs to the testa, i.e. against the total concentration gradient. This migration occurred only if the fermentation was sufficiently extensive to decrease the pH in the nib to <5.0, likely as a result of increased Cd mobility due to organic acid penetration into the nibs. The change in dry weight based nib Cd concentrations during fermentation was, on average, a factor 1.3 decrease. We propose that nib Cd can be reduced if the nib pH is sufficiently acidified during fermentation. However, a balance must be found between flavor development and Cd removal since extreme acidity is detrimental for cacao flavor.

Complementarity of MALDI and LA ICP mass spectrometry for platinum anticancer imaging in human tumor.

The follow-up of the Heated Intraoperative Chemotherapy (HIPEC) of peritoneal carcinomatosis would benefit from the monitoring of the penetration, distribution and metabolism of the drug within the tumor. As tumor nodules can be resected during the therapy, mass spectrometry imaging is a suitable tool for the evaluation of treatment efficacy, and, as a result, the therapy can be re-optimized. In this work we demonstrate the complementarity of laser ablation (LA) ICP mass spectrometry and MALDI imaging to study the penetration and distribution of two Pt-based metallodrugs (cisplatin and oxaliplatin) in human tumor samples removed from patients diagnosed with colorectal or ovarian peritoneal carcinomatosis. LA ICP MS offered sensitive (LOD for (195)Pt 4.8 pg s(-1)) imaging of platinum quasi-independently of the original species and the sample matrix and thus an ultimate way of verifying the penetration of the Pt-containing drug or its moieties into the tumor. MALDI imaging was found to suffer in some cases from signal suppression by the matrix leading to false negatives. In the case of the oxaliplatin metallodrug, the results obtained from ICP and MALDI MS imaging were coherent whereas in the case of cisplatin, species detected by ICP MS imaging could not be validated by MALDI MS. The study is the first application of the dual ICP and MALDI MS imaging to the follow-up of metallodrugs in human tumors.

Speciation analysis for trace levels of selenoproteins in cultured human cells.

A semi-quantitative method was developed for the non-targeted detection of trace levels of human selenoproteins in cytoplasmic cell extracts without the use of radioactive isotopes. The method was based on the direct detection of selenoproteins in iso-electrofocusing (IEF) electrophoretic strips by laser ablation-inductively coupled plasma mass spectrometry (LA-ICP MS). The proteins were identified in the non-ablated parts of the gel corresponding to the LA-ICP MS peak apexes by electrospray Orbitrap MS/MS. The method allowed a high resolution of the selenoproteins (peak width 0.06pH unit) using 3-10 pI strips. The protein detection limits were down to 1ngmL(-1) (as Se). The method was applied to the selenoprotein speciation in different human cell lines: Hek293 (kidney), HepG2 (liver), HaCaT (skin) and LNCaP (prostate). The principal proteins found included Selenoprotein 15 (Sep15), Glutathione peroxidase 1 (GPx1) and Glutathione peroxidase 4 (GPx4) and Thioredoxin reductase 1 (TRxR1) and Thioredoxin reductase 2 (TRxR2). BIOLOGICAL SIGNIFICANCE: Our paper presents the development of a semi-quantitative method for the non-targeted detection of trace levels of human selenoproteins in cytoplasmic cell extracts; it offers a first comprehensive screening of the entire biological selenoproteomes expressed in cell lines without the use of radioactive (75)Se. The method was based on the direct detection of selenoproteins in iso-electrofocusing (IEF) electrophoretic strips by laser ablation-inductively coupled plasma mass spectrometry (LA-ICP MS). The proteins were identified in the non-ablated parts of the gel corresponding to the LA-ICP MS peak apexes by electrospray Orbitrap MS/MS. The method allowed a high resolution of the selenoproteins (peak width 0.06pH unit) using 3-10 pI strips. The protein detection limits were down to 1ngmL(-1) (as Se); by far the lowest ever reported. The method was applied to the selenoprotein speciation in different human cell lines: Hek293 (kidney), HepG2 (liver), HaCaT (skin) and LNCaP (prostate). The principal proteins found included Selenoprotein 15 (Sep15), Glutathione peroxidase 1 (GPx1) and Glutathione peroxidase 4 (GPx4) and Thioredoxin reductase 1 (TRxR1) and Thioredoxin reductase 2 (TRxR2). The IEF-LA-ICPMS indicates the presence of multiple forms of some selenoproteins which are for the moment impossible to distinguish because of the similarity of the bottom-up, proteomics data sets.

Hemoglobin as a major binding protein for methylmercury in white-sided dolphin liver.

As methylmercury (MeHg) can be bioaccumulated and biomagnified in the trophic web, its toxicity for marine mammals is of major concern. Mercury speciation in marine biota has been widely studied, mainly focused on the discrimination and quantification of inorganic Hg and MeHg. Less attention has been paid to the interactions of Hg with biomolecules and the characterization of its specific binding, which play a key role in metabolic pathways controlling its uptake, transformation, and toxicity. In the studied white-sided dolphin (Lagenorhynchus acutus) liver homogenate (QC04LH4) sample, approximately 60% of the total MeHg was found in the water soluble fraction, specifically associated with high molecular weight biomolecules. The identity of the involved proteins was investigated (after tryptic digestion of the fraction) by µRPLC with parallel detection by ICP-MS and ESI-MS/MS. Molecular mass spectrometry experiments were carried out at high resolution (100000) to ensure accurate protein identification and determination of the MeHg binding sites. Cysteine residue on the dolphin hemoglobin ß chain was found to be the main MeHg binding site, suggesting that hemoglobin is a major MeHg binding protein in this marine mammal and could be a potential carrier of this MeHg from blood to liver prior to its degradation in this organ. In parallel, a significant proportion of selenium was found to be present as selenoneine and a potential role for this compound in Hg detoxification is discussed.

Inductively-coupled plasma mass spectrometry in proteomics, metabolomics and metallomics studies.

The potential of inductively-coupled plasma mass spectrometry (ICP-MS) and its complementarity to soft- ionization MS techniques are discussed in the context of the analysis for biomolecules. ICP-MS offers detection limits in the attomolar range, regardless of the molecular environment of the target element. The sensitivity is hardly affected by the sample matrix, chromatographic mobile phase, or co-eluted compounds. The abundance sensitivity over six decades and the linear dynamic range over nine decades make simultaneous multi-isotopic analysis routinely possible. The manuscript discusses the state-of-the-art of ICP-MS for the detection of proteins in gel electrophoresis and of peptides in 2D high-performance liquid chromatography. The possibilities of quantification to the degree of some post-translational modifications are highlighted. Attention is also paid to the role of ICP-MS in protein quantification via metal-coded labeling and to the use of differentially-labeled antibodies for the multiplexed biomarker analysis. The key role of ICP-MS in the emerging area of metallomics is briefly discussed.

A common highly conserved cadmium detoxification mechanism from bacteria to humans: heavy metal tolerance conferred by the ATP-binding cassette (ABC) transporter SpHMT1 requires glutathione but not metal-chelating phytochelatin peptides.

Cadmium poses a significant threat to human health due to its toxicity. In mammals and in bakers' yeast, cadmium is detoxified by ATP-binding cassette transporters after conjugation to glutathione. In fission yeast, phytochelatins constitute the co-substrate with cadmium for the transporter SpHMT1. In plants, a detoxification mechanism similar to the one in fission yeast is supposed, but the molecular nature of the transporter is still lacking. To investigate further the relationship between SpHMT1 and its co-substrate, we overexpressed the transporter in a Schizosaccharomyces pombe strain deleted for the phytochelatin synthase gene and heterologously in Saccharomyces cerevisiae and in Escherichia coli. In all organisms, overexpression of SpHMT1 conferred a markedly enhanced tolerance to cadmium but not to Sb(III), AgNO(3), As(III), As(V), CuSO(4), or HgCl(2). Abolishment of the catalytic activity by expression of SpHMT1(K623M) mutant suppressed the cadmium tolerance phenotype independently of the presence of phytochelatins. Depletion of the glutathione pool inhibited the SpHMT1 activity but not that of AtHMA4, a P-type ATPase, indicating that GSH is necessary for the SpHMT1-mediated cadmium resistance. In E. coli, SpHMT1 was targeted to the periplasmic membrane and led to an increased amount of cadmium in the periplasm. These results demonstrate that SpHMT1 confers cadmium tolerance in the absence of phytochelatins but depending on the presence of GSH and ATP. Our results challenge the dogma of the two separate cadmium detoxification pathways and demonstrate that a common highly conserved mechanism has been selected during the evolution from bacteria to humans.

Multitechnique mass-spectrometric approach for the detection of bovine glutathione peroxidase selenoprotein: focus on the selenopeptide.

Glutathione peroxidase (isolated from bovine erythrocytes) and its behaviour during alkylation and enzymatic digestion were studied by various hyphenated techniques: gel electrophoresis-laser ablation (LA) inductively coupled plasma (ICP) mass spectrometry (MS), size-exclusion liquid chromatography-ICP MS, capillary high-performance liquid chromatography (capHPLC)-ICP MS, matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) MS, electrospray MS, and nanoHPLC-electrospray ionization (ESI) MS/MS. ESI TOF MS and MALDI TOF MS allowed the determination of the molecular mass but could not confirm the presence of selenium in the protein. The purity of the protein with respect to selenium species could be evaluated by LA ICP MS and size-exclusion chromatography (SEC)-ICP MS under denaturating and nondenaturating conditions, respectively. SEC-ICP MS and capHPLC-ICP MS turned out to be valuable techniques to study the enzymolysis efficiency, miscleavage and artefact formation during derivatization and tryptic digestion. For the first time the parallel ICP MS and ESI MS/MS data are reported for the selenocysteine-containing peptide extracted from the gel; capHPLC-ICP MS allowed the sensitive detection of the selenopeptide regardless of the matrix and nanoHPLC-electrospray made possible its identification.