On-line Isotope Dilution Analysis Research Articles

SEC-ICP-MS and on-line isotope dilution analysis for characterisation and quantification of immunochemical assays.

This study presents a novel size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS) method for the characterisation and quantification of immunoassays with lanthanide-labelled antibodies. SEC-ICP-MS in combination with a double isotope dilution approach enabled facile validation of the antibodies' integrity, the determination of the batch to batch labelling efficiency, monitoring of each labelling step, and quantification of the immunocomplexes after incubation with the target protein. The addition of oxygen into the dynamic reaction cell improved the detection of sulphur as a marker for the antibodies and target protein via mass-shifting (LOD = 3.7ng/mL), whilst maintaining sufficient sensitivity for the analysis of the lanthanides. Ultra-high performance liquid chromatography (UHPLC) SEC ensured a rapid chromatographic method with separation times under 7min of the labelled and unlabelled antibodies, the immunocomplexes, and the unconjugated polymer used to lanthanide-label the antibodies. SEC calibration estimated the molecular weights of all peaks and provided valuable insights in immunochemical reactions and the stoichiometry of the reactants and products. A novel on-line isotope dilution analysis (IDA) enabled absolute quantification of sulphur and lanthanide signals and the protein of interest. The chromatographic separation of immunocomplexes and labelled antibodies allowed the simultaneous determination of the antibody/metal stoichiometry and target protein concentration from a single mass flow chromatogram. An immunoglobulin protein was quantified after incubation with an 153Eu-labelled primary polyclonal antibody. The procedure was validated with direct labelling of the target protein with 156Gd for parallel, simultaneous quantification. The concentration determined via direct labelling of the protein deviated 1.9% from the immunochemical approach employing 153Eu-labelled polyclonal antibodies. Graphical abstract.

On-line reverse isotope dilution analysis for spatial quantification of elemental labels used in immunohistochemical assisted imaging mass spectrometry via LA-ICP-MS

We present a novel on-line isotope dilution analysis (IDA) approach for the quantification of isotopically enriched metal labels used in immunohistochemical assisted imaging mass spectrometry.

Quantitative Bioimaging of Platinum via Online Isotope Dilution-Laser Ablation-Inductively Coupled Plasma Mass Spectrometry.

A new calibration strategy for elemental bioimaging based on online isotope dilution analysis (IDA) and laser ablation-inductively coupled plasma mass spectrometry (LA-ICPMS) was developed and applied for the quantification of platinum in rat kidney tissues. A dry 194Pt spike aerosol was added in a post-cell setup, and the natural 194Pt/195Pt isotope ratio of the sample aerosol from laser ablation was changed accordingly. Spike mass flow determination was carried out based on reversed IDA using a reference standard. Quantitative data obtained by the new approach correlated well with those obtained by external calibration when analyzing parallel tissue slices of rat kidney from cisplatin perfusion studies. The novel quantification approach is traceable to SI units, as IDA is an definitive method. Signal drifts are compensated as the second isotope acts as an internal standard.

Elemental Mass Spectrometry for Absolute Intact Protein Quantification without Protein-Specific Standards: Application to Snake Venomics.

Absolute protein quantification methods based on molecular mass spectrometry usually require stable isotope-labeled analogous standards for each target protein or peptide under study, which in turn must be certified using natural standards. In this work, we report a direct and accurate methodology based on capLC-ICP-QQQ and online isotope dilution analysis for the absolute and sensitive quantification of intact proteins. The combination of the postcolumn addition of 34S and a generic S-containing internal standard spiked to the sample provides full compound independent detector response and thus protein quantification without the need for specific standards. Quantitative recoveries, using a chromatographic core-shell C4 column for the various protein species assayed were obtained (96-100%). Thus, the proposed strategy enables the accurate quantification of proteins even if no specific standards are available for them. In addition, to the best of our knowledge, we obtained the lowest detection limits reported in the quantitative analysis of intact proteins by direct measurement of sulfur with ICPMS (358 fmol) and protein (ranging from 7 to 15 fmol depending on the assayed protein). The quantitative results for individual and simple mixtures of model proteins were statistically indistinguishable from the manufacturer's values. Finally, the suitability of the strategy for real sample analysis (including quantitative protein recovery from the column) was illustrated for the individual absolute quantification of the proteins and whole protein content in a venom sample. Parallel capLC-ESI-QTOF analysis was employed to identify the proteins, a prerequisite to translate the mass of quantified S for each chromatographic peak into individual protein mass.

Application of metallomic and metabolomic approaches in exposure experiments on laboratory mice for environmental metal toxicity assessment

Metals have a central role in biological systems, regulating numerous cellular processes, and in other cases having toxic or deleterious effects on the metabolism. Hence, the study of metal-induced changes in cellular metabolic pathways is crucial to understanding the biological response associated with environmental issues. In this context, the finding of biomarkers has great interest, representing -omics techniques, such as metallomics and metabolomics, powerful tools for this purpose. The present work evaluates the exposure of mice Mus musculus to toxic metals (As, Cd and Hg), considering the changes induced in both the metallome and metabolome as a consequence of the high genetic homology between Mus musculus/Mus spretus mice, which allows the use of the database from M. musculus to identify the proteins and metabolites expressed by M. spretus. For this purpose a metallomic approach based on size exclusion chromatography (SEC) in combination with other complementary orthogonal separation techniques and heteroelement monitoring by ICP-ORS-qMS was performed, followed by identification of metallobiomolecules by organic mass spectrometry. In addition, simultaneous speciation of selenoproteins and selenometabolites in mouse plasma was accomplished by tandem (double) SEC-(dual) affinity chromatography (AF)-HPLC and online isotope dilution analysis (IDA)-ICP-ORS-qMS. Finally, the simultaneous changes in metabolic expression in mice caused by metal exposure (metabolome) were considered, using direct infusion mass spectrometry (DI-ESI-QqQ-TOF-MS) of extracts from mice plasma. Subsequently altered metabolites were identified using MS/MS experiments. The results obtained under controlled conditions were extrapolated to homologous free-living mice captured in Doñana National Park (DNP) and surroundings (southwest Spain) affected by As, Cd and Hg pollution. In summary, such studies are needed to understand the effect of heavy metal exposure and cope with heavy metal toxicity.

Interfacing reversed-phase nanoHPLC with ICP-MS and on-line isotope dilution analysis for the accurate quantification of selenium-containing peptides in protein tryptic digests

An interface between nanoHPLC and ICP-MS was developed. It allowed the stable introduction into an ICP of mobile phases containing up to 90% of acetonitrile at flow rates of less than 500 nL min−1. The on-line post-column addition of an isotopically enriched spike at flow rates of less than 4 μL min−1 enabled isotope dilution quantification of heteroatom containing analytes while the consumption of the labelled isotope was low. The coupled system was applied to the accurate, sensitive and specific determination of selenopeptides in nanolitre volumes (11 nL) of a tryptic digest of selenomethionyl calmodulin (17002 Da). The peptides were separated by reversed phase nanoLC (340 nL min−1 flow rate) whereas ICP collision cell MS was used for the simultaneous detection of 80Se (analyte) and 76Se (spike). The absolute detection limit was 40 fg (80Se), a factor of 2 less than ever reported for a capillary HPLC-ICP-MS coupling. The sensitivity was constant during the chromatogram, regardless of the percentage of acetonitrile in the mobile phase. The selenium recovery was 103 ± 4%. For selenopeptide analysis the sum of Se determined in each of the peaks equalled the total Se injected on the column. Since the tryptic peptides, miscleaved and/or oxidized peptides, incompletely digested protein and undigested protein could be determined in one run, the method allowed the precise evaluation of the efficiency and quality of tryptic digestion using several nanolitres of sample only.

Automated On-Line Isotope Dilution Analysis with ICP-MS Using Sandwich Flow Injection

An automated flow injection (FI) manifold is described to perform the addition of isotopic spikes to aqueous samples on-line with ICP-MS for isotope dilution (ID) analysis. The manifold uses the sandwich technique (with the nested loop approach) to perform an injection of the isotopic spike solution within a sample (or standard) plug, the resulting sample-spike-sample sequence being pushed toward the nebulizer by a 1% HNO(3) carrier. A standard, which must contain one element not present in the spike solution to allow the determination of the dispersion coefficient, must also be used to allow a reverse isotope dilution analysis, as well as corrections for mass discrimination and/or spectroscopic interferences. Indeed, because the signals from the individual isotopes are monitored continuously, only one isotope free of spectroscopic interference is required for elements whose isotopic distribution does not vary in nature (two isotopes are still needed for the other elements), as a correction for the interference can be made by comparison with the signals from the standard. Furthermore, this automated approach makes ID-ICP-MS a faster method and does not require any preliminary analysis of the sample because the concentration profile resulting from FI allows the selection of the best isotopic ratio. It was successfully applied to the determination of Mo in saline water.

On-Line Isotope Dilution Analysis with ICPMS Using Reverse Flow Injection

A simple flow injection manifold is described to perform the addition of isotopic spikes to aqueous samples on-line with ICPMS. The analysis involves one multielement spike injection in the sample ...