Evaluation of different isotope dilution mass spectrometry strategies for the characterization of naturally abundant and isotopically labelled peptide standards.

Natural abundance and isotopically labelled tryptic peptides are routinely employed as standards in quantitative proteomics. The certification of the peptide content is usually carried out by amino acid analysis using isotope dilution mass spectrometry (IDMS) after the acid hydrolysis of the peptide. For the validation and traceability of the amino acid analysis procedure, expensive certified peptides must be employed. In this work we evaluate different IDMS alternatives which will reduce the amount of certified peptide required for validation of the amino acid analysis procedure. In this context, the characterization of both natural and isotopically labelled synthetic angiotensin I peptides was carried out. First, we applied a fast procedure for peptide hydrolysis based on microwave-assisted digestion and employed two certified peptide reference materials SRM 998 angiotensin I and CRM 6901-b C-peptide for validation of the hydrolysis procedure. The amino acids proline, leucine, isoleucine, valine, tyrosine, arginine and phenylalanine were evaluated for their suitability for peptide certification by IDMS by both liquid chromatography with tandem mass spectrometry (LC-MS/MS) and gas chromatography with mass spectrometry (GC)-MS/MS. Then, natural angiotensin I and 13C1-labelled angiotensin I were synthesized in-house and purified by preparative liquid chromatography. The concentration of the 13C1-labelled angiotensin I peptide was established by reverse IDMS in its native form using SRM 998 angiotensin I as reference. The concentration of the natural synthesized peptide was determined by IDMS both using the 13C1-labelled peptide in its native form and by amino acid analysis showing comparable results. Finally, the synthetic naturally abundant angiotensin I peptide was employed as "in-house" standard for the validation of subsequent peptide characterization procedures. Therefore, the novelty of this work relies on, first, the development of a faster hydrolysis procedure assisted by focused microwaves, providing complete hydrolysis in 150 min, and secondly, a validation strategy combining GC-MS and LC-MS/MS that allowed us to certify the purity of an in-house-synthesized peptide standard that can be employed as quality control in further experiments.

Determination of 3-monoiodotyrosine and 3,5-diiodotyrosine in newborn urine and dried urine spots by isotope dilution tandem mass spectrometry.

High levels of 3-mono- and 3,5-diiodotyrosine (MIT and DIT, respectively) in urine have been related to iodotyrosine dehalogenase 1 deficiency, a type of congenital hypothyroidism. However, the determination of MIT and DIT in urine is not included in newborn screening programs performed in clinical laboratories to detect inborn errors of metabolism. We report here on the development of an analytical method for the determination of MIT and DIT in newborn urine and dried urine spots (DUS) by Liquid Chromatography Isotope Dilution tandem Mass Spectrometry (LC-IDMSMS). The development included the synthesis of 15N-monoiodotyrosine and 13C2-diiodotyrosine through the iodination of 15N-tyrosine and 13C2-tyrosine, respectively, using bis(pyridine)iodonium(I) tetrafluoroborate (IPy2BF4). Both labelled analogues were added at the beginning of the sample preparation procedure and used to develop both single- and double-spike LC-IDMS methods for the determination of MIT and DIT. The developed double spike methodology was able to quantify and correct possible MIT ↔ DIT interconversions throughout the sample preparation, which was observed for concentrated urine samples but not for DUS. Suppression matrix effects on the absolute signals of MIT and DIT were observed in urine samples but did not affect the IDMS results as recoveries on urine samples at different dilution factors could be considered quantitative. Method detection limits were 0.018 and 0.046 ng g-1 (limits of quantification 0.06 and 0.15 ng g-1) by single-spike IDMS, for MIT and DIT, respectively, in the analysis of urine samples and 0.07 and 0.05 ng g-1 (limits of quantification 0.23 and 0.17 ng g-1) for MIT and DIT, respectively, in the analysis of DUS. No significant differences were obtained for MIT concentrations in the analysis of the same newborn samples stored as liquid urine or DUS when the results were corrected for the creatinine content. Finally, 36 DUS samples from healthy newborns were analyzed and MIT was detected in all samples at low ng mg-1creatinine levels.

Anion-Specific Sulfur Isotope Analysis by Liquid Chromatography Coupled to Multicollector ICPMS.

An accurate method has been developed to measure, in a single analytical run, δ34S in sulfite, sulfate and thiosulfate in water samples by liquid chromatography combined with multicollector inductively coupled plasma mass spectrometry (MC-ICPMS). The method is based on the anionic exchange separation of sulfur species prior to their online isotope ratio determination by MC-ICPMS. Mass bias correction was accomplished by a novel approach based on the addition of an internal sulfur-containing standard to the sample. This innovative approach was compared to the sample-standard bracketing procedure. On-column isotopic fractionation was observed and therefore corrected by external calibration. Isotopic ratios were calculated by linear regression slope (LRS), an advantageous method for transient signals, leading to a combined uncertainty of δ34S below 0.25‰ and a reproducibility below 0.5‰ for the injection of 1 µg of S. The method was successfully applied to the measurement of δ34S in synthetic solutions and environmental water samples. Matrix effects leading to δ34S overestimation were observed for sulfate in some samples with high sodium/sulfate mass ratios. The developed analytical procedure simplifies the δ34S analysis of liquid environmental samples since preparation steps are no longer required and allows the analysis of several sulfur-containing species in a single run.

Concentration of mercury species in hair, blood and urine of individuals occupationally exposed to gaseous elemental mercury in Asturias (Spain) and its comparison with individuals from a control group formed by close relatives.

Between November 19th, 2012 and December 3rd, 2012, 50 workers were intoxicated with gaseous Hg in San Juan de Nieva (Asturias, Spain) during the maintenance of a heat exchanger of a zinc manufacturer. We have quantified the concentration of methylmercury (MeHg), ethylmercury (EtHg) and Hg(II) in blood, hair and urine samples of those individuals taken three years after the accident. Blood, hair and urine of their closest relatives were also analyzed to assess whether the mercury burden present in the intoxicated individuals was due to the occupational exposure or to environmental or lifestyle-related factors. The determination of the mercury species in the samples was carried out applying multiple spiking Isotope Dilution GC-ICP-MS. This procedure corrects for possible interconversion reactions between the Hg species during the sample preparation procedure. Linear correlations were observed for both groups when plotting MeHg in blood vs MeHg in hair, and MeHg in hair vs Hg (II) in urine. The concentrations of Hg species in the intoxicated individuals were not significantly different from those obtained in the control group except for MeHg in blood. Significantly higher levels of MeHg in blood were obtained in some of the intoxicated individuals who had not consumed fish or seafood since the accident. A different correlation between MeHg in hair and MeHg in blood was obtained for these individuals compared to the control group who showed a hair-to-blood ratio consistent with the reported value for people exposed to Hg via fish consumption. Our results suggest that ingested MeHg followed the same pathway of deposition in hair in exposed and non-exposed individuals. However, the exposed individuals with high MeHg levels in blood showed a significantly different extent of MeHg deposition in hair compared to the control group.

Isotopically Enriched Tracers and Inductively Coupled Plasma Mass Spectrometry Methodologies to Study Zinc Supplementation in Single-Cells of Retinal Pigment Epithelium in Vitro.

Mass spectrometry-based techniques, such as inductively coupled plasma mass spectrometry (ICPMS) and laser ablation (LA) ICPMS, combined with an isotope pattern deconvolution mathematical tool are proposed for a better understanding of supplementation studies in cultured cells. An in vitro model of human retinal pigment epithelium (HRPEsv) cells was treated with different concentrations (0-150 µm Zn, 1 mL) of enriched stable isotope tracers of Zn in the form of sulfate and/or gluconate. Supplementations with t68ZnSO4 or t70Zn-gluconate alone and in combination (1:1 molar ratio) were investigated to evaluate the exogenous contribution and distribution of Zn in the treated cells. In order to obtain not only the Zn concentration for a cell population (mineralized cells) but also single cell information about the contribution of exogenous Zn and their distribution within micrometer cells structures, LA-ICPMS was employed to directly analyze cryopreserved cells. natZn, t68Zn, and t70Zn molar fraction images obtained from cells and cell aggregates allowed confirming the uptake of exogenous Zn by HRPEsv cells, being t68Zn and t70Zn molar fractions close to 1 in the cell nuclei. Under the selected experimental conditions tested (24 h treatments), no significant differences were obtained in the Zn distribution depending on its chemical form.

Quantitative Assessment of Individual Populations Present in Nanoparticle-Antibody Conjugate Mixtures Using AF4-ICP-MS/MS.

A current remaining challenge in nanotechnology is the fast and reliable determination of the ratios between engineered nanoparticles and the species attached to their surface after chemical functionalization. The approach proposed herein based on the online coupling of asymmetric flow field-flow fractionation (AF4) with inductively coupled plasma-tandem mass spectrometry (ICP-MS/MS) allows for the first time the direct determination of such ratios in CdSe/ZnS core-shell quantum dot:rat monoclonal IgG2a antibody (QD:Ab) conjugate mixtures in a single run without any previous sample preparation (i.e., derivatization). AF4 provides full recovery and adequate resolution of the resulting bioconjugate from the excess of nanoparticles and proteins used in the different bioconjugation mixtures (1:1, 2:1, and 3:1 QD:Ab molar ratios were assessed). The point-by-point determination by ICP-MS/MS of the metal to sulfur ratios along the bioconjugate fractographic peak allowed disclosing the mixture of the different species in the bioconjugated sample, providing not only the limits of the range of QD:Ab ratios in the different bioconjugate species resulting after functionalization but also a good estimation of their individual relative abundance in the mixture. Interestingly, a wide variety of compositions were observed for the different bioconjugate mixtures studied (QD:Ab molar ratios ranging from 0.27 to 4.6). The resulting weighted QD:Ab ratio computed in this way for each bioconjugate peak matches well with both the global (average) QD:Ab ratio experimentally obtained by the simpler peak area ratio computation and the theoretical QD:Ab molar ratios assayed, which internally validates the procedure developed.

Development of a Common Procedure for the Determination of Methylmercury, Ethylmercury, and Inorganic Mercury in Human Whole Blood, Hair, and Urine by Triple Spike Species-Specific Isotope Dilution Mass Spectrometry.

We report the first common methodology for the simultaneous determination of methylmercury (MeHg), ethylmercury (EtHg), and inorganic mercury (Hg(II)) in human blood hair and urine. With the exception of the initial sample mass (0.15 g for blood, 0.5 g for urine, and 0.1 g for hair), the same sample preparation and gas chromatography-inductively coupled plasma mass spectrometry (GC-ICPMS) measurement conditions are employed for the three matrixes providing experimental values in agreement with the certified values in the analysis of NIST SRM 955c (Caprine Blood) Level 3 and the certified human hairs IAEA 085 and IAEA 086. Also, the method provides quantitative recoveries for the three Hg species in the analysis of fortified human urine samples at 1, 2, and 5 ng Hg g-1. Mercury species concentrations for levels 2 and 4 of SRM 955c are reported here for the first time. A systematic interconversion of EtHg into Hg(II) was obtained for all matrixes reaching values up to 95% in blood, 29% in hair, and 11% in urine. MeHg dealkylation was also observed in a lesser extent in blood and hair analyses, but it was not observed when analyzing urine samples. Hg methylation was not observed in any matrix. The amount of NaBPr4 added for derivatization has been found to be the main factor responsible for Hg species interconversion. This work demonstrates for the first time that experimental conditions optimized for SRM 955c (caprine blood) are not valid for human blood samples as the optimum initial sample amount for a real sample is more than 3 times lower than that for SRM 955c.

Evaluation of sulfur isotopic enrichment of urine metabolites for the differentiation of healthy and prostate cancer mice after the administration of 34S labelled yeast.

Sulfur isotopic enrichment of urine metabolites in healthy and prostate cancer mice using 34S enriched yeast and High Performance Liquid Chromatography coupled to Multicollector Inductively Coupled Plasma Mass Spectrometry (HPLC-MC-ICP-MS) has been evaluated. A 30 weeks experiment (since the eleventh to the fortieth week of life) was carried out collecting the urine of three healthy mice and three transgenic mice with prostate cancer during 24h after a single oral administration of a 34S enriched yeast slurry. The isotopic enrichment of different sulphur metabolites was monitored by coupling a C18 reverse phase HPLC column with a multicollector ICP-MS using a membrane desolvating system. Quantification of sulfur in the chromatographic peaks was carried out by post-column isotope dilution using a 33S enriched spike. Differences between the 34S enrichment in the urine metabolites of healthy and prostate cancer mice were found from the beginning of the disease. Both populations could be differentiated using a principal component analysis (PCA). Finally, 7 unknown mice were correctly classified in each population using a linear discriminant analysis.

Potential of Nassarius nitidus for monitoring organotin pollution in the lagoon of Bizerta (northern Thunisia).

Imposex and butyltin burden were assessed in Nassarius nitidus, Bolinus brandaris and Hexaplex trunculus collected at five stations in the Bizerta lagoon. Biological analysis showed that imposex followed type (a) in N. nitidus (distal evolution), against type (d) in the two muricids (proximal evolution). Imposex indices were higher in sites located nearby sources of tributyltin and N. nitidus was the least affected species of the five sites, followed by B. brandaris and H. trunculus. Butyltin analysis showed lower accumulation in N. nitidus followed by H. trunculus and B. brandaris. This study has allowed the gathering of data on imposex in a snail studied for the first time in Tunisia (N. nitidus). It suggests the possibility of using such snail as a complementary species for organotin monitoring programs in the Mediterranean and further confirmed that H. trunculus is the most suitable species for such investigations.

Identification of a tri-iron(III), tri-citrate complex in the xylem sap of iron-deficient tomato resupplied with iron: new insights into plant iron long-distance transport.

The identification of Fe transport forms in plant xylem sap is crucial to the understanding of long-distance Fe transport processes in plants. Previous studies have proposed that Fe may be transported as an Fe-citrate complex in plant xylem sap, but such a complex has never been detected. In this study we report the first direct and unequivocal identification of a natural Fe complex in plant xylem sap. A tri-Fe(III), tri-citrate complex (Fe(3)Cit(3)) was found in the xylem sap of Fe-deficient tomato (Solanum lycopersicum Mill. cv. 'Tres Cantos') resupplied with Fe, by using an integrated mass spectrometry approach based on exact molecular mass, isotopic signature and Fe determination and retention time. This complex has been modeled as having an oxo-bridged tri-Fe core. A second complex, a di-Fe(III), di-citrate complex was also detected in Fe-citrate standards along with Fe(3)Cit(3), with the allocation of Fe between the two complexes depending on the Fe to citrate ratio. These results provide evidence for Fe-citrate complex xylem transport in plants. The consequences for the role of Fe to citrate ratio in long-distance transport of Fe in xylem are also discussed.