Development of an ICP-MS/MS approach for absolute quantification and determination of phosphodiester to phosphorothioate ratio in therapeutic oligonucleotides.

A new analytical method based on ICP-MS/MS is proposed for the characterization of synthetic phosphorothioate oligonucleotides. Absolute quantification of oligonucleotides is challenging, as well as the determination of phosphodiester to phosphorothioate ratio for phosphorothioate oligonucleotides. Both are considered as critical quality attributes and should be determined using robust validated methods. The method we developed was designed to be easy to apply, fast, and robust. It allows simultaneous absolute quantification of an oligonucleotide (based on the quantification of phosphorus), determination of the phosphodiester to phosphorothioate ratio (based on the quantification of phosphorus and sulfur) and optionally determination of sodium (or any other metal) as a counter ion. The performance of the method was demonstrated on O,O-diethyl thiophosphate potassium salt, a well characterized model substance that possesses similar composition to phosphorothioate oligonucleotides. Method was also tested on different synthetic phophorothioate oligonucleotides, showing excellent accuracy and precision.

Selenium-regulated hierarchy of human selenoproteome in cancerous and immortalized cells lines.

BACKGROUND: Selenoproteins (25 genes in human) co-translationally incorporate selenocysteine using a UGA codon, normally used as a stop signal. The human selenoproteome is primarily regulated by selenium bioavailability with a tissue-specific hierarchy. METHODS: We investigated the hierarchy of selenoprotein expression in response to selenium concentration variation in four cell lines originating from kidney (HEK293, immortalized), prostate (LNCaP, cancer), skin (HaCaT, immortalized) and liver (HepG2, cancer), using complementary analytical methods. We performed (i) enzymatic activity, (ii) RT-qPCR, (iii) immuno-detection, (iv) selenium-specific mass spectrometric detection after non-radioactive 76Se labeling of selenoproteins, and (v) luciferase-based reporter constructs in various cell extracts. RESULTS: We characterized cell-line specific alterations of the selenoproteome in response to selenium variation that, in most of the cases, resulted from a translational control of gene expression. We established that UGA-selenocysteine recoding efficiency, which depends on the nature of the SECIS element, dictates the response to selenium variation. CONCLUSIONS: We characterized that selenoprotein hierarchy is cell-line specific with conserved features. This analysis should be done prior to any experiments in a novel cell line. GENERAL SIGNIFICANCE: We reported a strategy based on complementary methods to evaluate selenoproteome regulation in human cells in culture.

An insight into silver nanoparticles bioavailability in rats.

A comprehensive study of the bioavailability of orally administered silver nanoparticles (AgNPs) was carried out using a rat model. The silver uptake was monitored in liver and kidney tissues, as well as in urine and in feces. Significant accumulation of silver was found in both organs, the liver being the principal target of AgNPs. A significant (∼50%) fraction of silver was found in feces whereas the fraction excreted via urine was negligible (< 0.01%). Intact silver nanoparticles were found in feces by asymmetric flow field-flow fractionation (AsFlFFF) coupled with UV-Vis analysis. Laser ablation-ICP MS imaging showed that AgNPs were able to penetrate into the liver, in contrast to kidneys where they were retained in the cortex. Silver speciation analysis in cytosols from kidneys showed the metallothionein complex as the major species whereas in the liver the majority of silver was bound to high-molecular (70-25 kDa) proteins. These findings demonstrate the presence of Ag(i), released by the oxidation of AgNPs in the biological environment.

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.

Large-scale speciation of selenium in rice proteins using ICP-MS assisted electrospray MS/MS proteomics.

A Se-targeted bottom-up proteomics approach was developed for the identification of Se-containing proteins in rice grown naturally on seleniferous soils. The proteins were separated by 2D gel electrophoresis. The position of Se-containing spots was tentatively identified by the correlation between the 1D isoelectrofocusing (IEF) and 1D SDS electropherograms of a sample aliquot and confirmed by (78)Se imaging in the 2D gel. The method was complemented by the ICP-MS assisted shotgun proteomics approach. The proteins were identified by capHPLC with the dual ICP MS and electrospray Orbitrap MS detection. The first ever comprehensive study of rice selenoproteome revealed the presence of selenium, as both selenomethionine (SeMet) and selenocysteine (SeCys) residues, in a dozen proteins including a 19 kDa globulin, granule-bound starch synthase, and the family of glutelin-type seed storage proteins.

A comparative study of the Se/S substitution in methionine and cysteine in Se-enriched yeast using an inductively coupled plasma mass spectrometry (ICP MS)-assisted proteomics approach.

A proteomics approach based on 2D gel electrophoresis followed by HPLC-electrospray Orbitrap MS/MS was developed to investigate the replacement and the degree of the Se/S substitution in methionine and cysteine in Se-rich yeast. Capillary HPLC-inductively coupled plasma mass spectrometry (ICP-MS), employed in parallel to capHPLC-ESI MS, indicated the virtual independence of the ESI MS response of the peptide structure (in the elution range of 30-65% methanol), and hence, the use of ESI MS data to determine the SeCys/Cys and SeMet/Met substitution ratios. For the first time a considerable incorporation of selenocysteine (SeCys) in proteins of the yeast proteome despite the absence of the UGA codon was demonstrated. The SeMet/Met and SeCys/Cys ratios were determined in a large number of peptides (57 and 26, respectively) issued from the tryptic digestion of 19 Se-containing proteins located in the gel by laser ablation-ICP MS imaging. The average Se/S substitution in methionine was 42.9±35.0 and was protein dependent with ratios ranging from 5 to 160 for individual peptides. The substitution of sulphur in cysteine (14.1±4.8%) in the cysteine-containing peptides was relatively similar (ratios from 9 to 23). Taking into account that the cysteine/methionine average ratio (2:1) in the yeast protein fraction, the study allowed the conclusion that 10-15% of selenium present in Se-enriched yeast is in the form of selenocysteine making up the mass balance of selenium species. BIOLOGICAL SIGNIFICANCE: For the first time a considerable incorporation of selenocysteine (SeCys) in proteins of the yeast proteome despite the absence of the UGA codon was demonstrated. It was achieved using a proteomics approach based on 2D gel electrophoresis followed by HPLC-electrospray Orbitrap MS/MS in order to investigate the replacement and the degree of the Se/S substitution in methionine and cysteine in Se-rich yeast.

Privileged incorporation of selenium as selenocysteine in Lactobacillus reuteri proteins demonstrated by selenium-specific imaging and proteomics.

An analytical approach was developed to study the incorporation of selenium (Se), an important trace element involved in the protection of cells from oxidative stress, into the well-known probiotic Lactobacillus reuteri Lb2 BM-DSM 16143. The analyses revealed that about half of the internalized Se was covalently incorporated into soluble proteins. Se-enriched proteins were detected in 2D gels by laser ablation inductively coupled plasma mass spectrometry imaging (LA-ICP MSI) and identified by capillary HPLC with the parallel ICP MS ((78)Se) and electrospray Orbitrap MS/MS detection. On the basis of the identification of 10 richest in selenium proteins, it was demonstrated that selenium was incorporated by the strain exclusively as selenocysteine. Also, the exact location of selenocysteine within the primary sequence was determined. This finding is in a striking contrast to another common nutraceutical, Se-enriched yeast, which incorporates Se principally as selenomethionine.

Characterization of selenium incorporation into wheat proteins by two-dimensional gel electrophoresis-laser ablation ICP MS followed by capillary HPLC-ICP MS and electrospray linear trap quadrupole Orbitrap MS.

A method has been developed for a rapid and precise location of selenium-containing proteins in large two-dimensional (2D) electrophoresis gels. A sample was divided into four aliquots which were analyzed in parallel by 1D isoelectric focusing electrophoresis (IEF)-laser ablation (LA) inductively coupled plasma mass spectrometry (ICP MS), 1D sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE)-LA ICP MS, and, in duplicate, by 2D IEF-PAGE. On the basis of the 1 D electropherograms obtained, areas supposed to contain the largest concentrations of Se were subjected to LA ICP MS imaging to locate precisely the position of Se-containing proteins which were then identified in the parallel 2D gel by electrospray Orbitrap MS/MS. The method was applied to the identification and semiquantitative determination of selenium storage proteins in wheat. MS evidence is presented for the Se-S substitution in plants not only in methionine but also in cysteine.

Probing of bismuth antiulcer drug targets in H. pylori by laser ablation-inductively coupled plasma mass spectrometry.

A method that allows partial denaturation of protein ligands in Bi- and Zn-protein complexes, leaving the metal coordination centre intact, was developed. It was based on the reduction of the S-S bridges with tris(2-carboxyl)phosphine followed by derivatization with iodoacetamide. Consequently conditions that allow the separation of Bi- and Zn-protein complexes using SDS electrophoresis were found. The separation efficiency was much higher than that in non-denaturating blue native electrophoresis. The method allowed the detection of seven Bi-binding protein candidates in H. pylori treated with bismuth subcitrate, some of which-fructose-bisphosphate aldolase (33.6 kDa), urease alpha subunit (26.4 kDa), and the 16.8 kDa proteins: 30S ribosomal protein S6 and neutrophil activating protein (NapA)-were bio-induced during the treatment. The method also allowed the monitoring of the changes in the Zn-proteome during treatment of H. pylori with the Bi-drug, which was found to increase the concentration of the Zn-binding proteins with particularly strong expression of the urease, S-adenosylmethionine synthetase and the above 16.8 kDa proteins.