Jorge Ruiz Encinar

Triple Quad ICPMS (ICPQQQ) as a New Tool for Absolute Quantitative Proteomics and Phosphoproteomics

It is clear that sensitive and interference-free quantification of ICP-detectable elements naturally present in proteins will boost the role of ICPMS in proteomics. In this study, a completely new way of polyatomic interference removal in ICPMS for detection of sulfur (present in the majority of proteins as methionine or cysteine) and phosphorus (present in phosphorylated proteins) is presented. It is based on the concept of tandem mass spectrometry (QQQ) typically used in molecular MS. Briefly, the first quadrupole can be operated as 1 amu window band-pass mass filter to select target analyte ions ((31)P, (32)S, and their on-mass polyatomic interferences). In this way, only selected ions enter the cell and react with O(2), reducing the interferences produced by matrix ions as well as background noise. After optimization of the cell conditions, product ions formed for the targets, (47)PO(+) and (48)SO(+), could be detected with enhanced sensitivity and selectivity. The coupling to capillary HPLC allowed analysis of S- and P-containing species with the lowest detection limits ever published (11 and 6.6 fmol, respectively). The potential of the approach for proteomics studies was demonstrated for the highly sensitive simultaneous absolute quantification of different S-containing peptides and phosphopeptides.

Calcification rate and temperature effects on Sr partitioning in coccoliths of multiple species of coccolithophorids in culture

Five common placolith-bearing coccolithophorid algae—Gephyrocapsa oceanica, Coccolithus pelagicus, Calcidiscus leptoporus, Umbilicosphaera sibogae (var. sibogae and var. foliosa), and Emiliania huxleyi—were cultured to investigate controls on Sr partitioning in coccolith calcite. For identical temperature and media composition, Sr partitioning varies by more than 30% in exponential phase cultures of the five species and is linearly related to rates of calcite production/cell (ρ=0.91). Exponential phase culture experiments with three strains of C. leptoporus and six strains of G. oceanica at varying temperatures show variations in Sr partitioning of 20% and 30%, respectively. With C. leptoporus, Sr partitioning is equally correlated with temperature and calcification rate (ρ=0.8), which themselves are highly correlated; the slope of the relationship between DSr and calcification rate is comparable to that observed in all species at constant temperature. However, in G. oceanica, increased temperature appears to enhance Sr incorporation by up to 2% to 1.6% °C−1 in the range of 15 to 30 °C. The strong influence of calcification rate on Sr partitioning may be useful for inferring past variations in coccolithophorid productivity from Sr partitioning in coccolith sediments if the influence of temperature on Sr partitioning can be resolved. Because the relationship between calcite production and Sr partitioning is linear, a proportional change in calcification should be expressed much more strongly in the Sr/Ca ratio of large species with rapid calcite production than in smaller species, which produce calcite more slowly. Consequently, it may be possible to separate temperature and calcification influences on coccolith Sr/Ca by separately analyzing Sr/Ca in species that produce calcite rapidly and those that produce calcite slowly, if both undergo comparable relative changes in calcification rates.

A comparison between quadrupole, double focusingand multicollector ICP-MS instruments

A quadrupole-based instrument (HP-4500), a double focusing single collector instrument (Element) and a single focusing multicollector (IsoProbe) were all evaluated for the precise and accurate measurement of lead isotope ratios using Tl as internal isotope ratio standard. The isotope ratio accuracy was evaluated using NIST 981 Common Lead Isotope standard reference material and an enriched 204Pb spike. Mass bias correction was performed using an exponential model in all the three instruments and equations for the evaluation of the total combined uncertainty arising from the correction for the dead time and mass bias were developed. It was observed that, in the correction for mass bias, the uncertainty in the natural Tl ratio was the main source of uncertainty for the multicollector instrument while, for the quadrupole and double focusing single collector instruments, the measured isotope ratio was the main source of uncertainty.

Absolute and site-specific quantification of protein phosphorylation using integrated elemental and molecular mass spectrometry: its potential to assess phosphopeptide enrichment procedures.

The validity of using elemental phosphorus standards to accurately and precisely quantify phosphopeptides by capillary HPLC (capHPLC) coupled to ICP-collison cell-MS is investigated in detail. Operating requirements to maintain stable (31)P sensitivity along the reversed-phase gradient are described. Specifically, the use of an optimum postcolumn makeup flow with a defined acetonitrile content turned out to be necessary to buffer the acetonitrile variation of the capillary chromatographic eluent and ensure plasma stability. Then, a highly pure P-containing standard (bis(4-nitro-phenyl) phosphate, BNPP) was spiked into the samples and used to quantify them with very low absolute errors (2-4%) and excellent precision (3-6%). The capHPLC-ICPMS method showed excellent linearity over 3 orders of magnitude and provided adequate detection limits (110 fmol, 3.4 pg P). Accurate quantification of the phosphopeptides present in a tryptic digest of beta-casein and casein from bovine milk was then attempted. Previously, and in order to be able to close mass balances, total P contents, percentages of inorganic P present, and recoveries from the reversed-phase column used in the separation were computed for each sample. Quantification using the spiked BNPP for the different phosphopeptides detected matched the expected values well validating the quantitative methodology proposed. The capHPLC-ESIMS analysis allowed elucidating amino acid sequences, a requisite still necessary to translate the determined amount of P in each chromatographic peak into amount of phosphopeptide. The great potential of these strategies, based on ICPMS detection, to assess the many procedures proposed and commonly used for purification, preconcentration, and/or isolation of phosphopeptides in phosphoproteomics studies is demonstrated using a commercially available titanium dioxide (TiO(2)) cartridge for phosphopeptide enrichment from complex mixtures. Quantitative results obtained allow one to assess individual phosphopeptide recoveries from the TiO(2) cartridge with unsurpassed accuracy. Of course, this information is essential for reliable absolute quantifications in phosphoproteomics.

Capillary HPLC-ICPMS and Tyrosine Iodination for the Absolute Quantification of Peptides Using Generic Standards

The validity of using tyrosine iodination chemistry for the absolute and generic quantification of peptides by capillary high-performance liquid chromatography (capHPLC) coupled to inductively coupled plasma mass spectrometry (ICPMS) is investigated in detail. In this approach, two iodine atoms are specifically bioconjugated to the meta positions of the aromatic ring of every tyrosine residue. Characterization studies by capHPLC with parallel ICPMS and electrospray ionization tandem mass spectrometry (ESIMS/MS) detection clearly showed that such labeling iodination reaction affords one to obtain most accurate peptide determinations (after translation of the picomoles of iodine, quantified by ICPMS in each chromatographic peak, into picomoles of the corresponding labeled peptide). It is demonstrated that only, but every, tyrosine residue present in the peptide is completely diiodinated. The excellent detection limits for iodine using ICPMS allowed robust and highly sensitive tyrosine-containing peptide quantification (480 pM, 480 amol absolute). Derivatization is easily accomplished in a water/acetonitrile solution in only 2 min. Moreover, since the signal in ICPMS is completely independent from the chemical species containing the detected element, any iodine-containing standard (e.g., iodobenzoic acid) could be used as internal standard for the absolute quantification of every iodine-labeled tyrosine-containing peptide separated and detected along the gradient. The approach was optimized for tyrosine labeling and then validated by application to the absolute quantification of the three standard peptides present in the only reference material for peptide quantity (NIST 8327) commercially available. Identification of the species quantified by ICPMS was carried out by parallel capHPLC-ESI quadrupole time-of-flight (Q/TOF) analysis and corresponded, as expected, to the diiodinated peptides. The collision-induced dissociation (CID) spectra obtained demonstrated unequivocally the specific and complete derivatization of the tyrosine residues. The obtained quantitative results closely matched the reference values reported by the National Institute of Standards and Technology (NIST). In terms of precision, the relative standard deviation was as low as 3% RSD. Finally the approach was tested for the absolute quantification of proteins using a model standard protein (beta-casein). Results agreed again with the value specified showing that this labeling reaction is compatible with tryptic digestion.

Determination of cadmium in environmental and biological reference materials using isotope dilution analysis with a double focusing ICP-MS: a comparison with quadrupole ICP-MS

A detailed examination of the performance of a double focusing (DF) sector field ICP-MS (Element, Finnigan MAT) for the ID-ICP-MS determination of cadmium in biological and environmental samples is described and compared with a quadrupole-based instrument (HP4500, Hewlett Packard). Experimental parameters governing the instrumental precision and accuracy for isotope ratio measurements of cadmium in the DF-ICP-MS are first characterized. Systematic errors, including dead time, mass bias effects and spectroscopic interferences, can be easily corrected in the two instruments studied. Detector dead time has only to be taken into account using the DF-ICP-MS. Typical isotope ratio precisions of 0.2-0.3% can be obtained routinely with both instruments. However, the precision of the measurements in the DF-ICP-MS can be improved (<0.1%) by increasing the number of scans. Moreover, for very low cadmium concentrations the DF-ICP-MS at low resolution setting (R=300) can provide better isotope ratio precision than the quadrupole ICP-MS because of its higher sensitivity. Both ICP-MS instruments were successfully applied to the determination of very low levels of cadmium by ID-ICP-MS (by both conventional and on-line ID modes) in biological and environmental certified reference materials, including Freeze-Dried Urine NIST SRM-2670, Horse Kidney IAEA H-8, Lichens BCR TP-25, Marine Sediment PACS-1 and Riverine Water SLRS-3. Combined uncertainty associated with the determinations is addressed.

Determination of butyltin compounds in coastal sea-water samples using isotope dilution GC-ICP-MS

A rapid, accurate, sensitive and simple method for the simultaneous determination of mono-, di- and tributyltin (MBT, DBT and TBT, respectively) in coastal sea-water samples was developed. The method is based on the application of isotope dilution methodologies using a 119Sn enriched spike containing all three butyltin compounds. Sample volumes of 100 ml were introduced in pre-cleaned glass volumetric flasks and spiked with the mixed enriched spike. After 15 min equilibration, sodium tetraethylborate, acetate buffer of pH 5.4 and 1 ml of hexane were added and the mixture shaken for 10 min. The hexane phase was recovered, evaporated to near dryness under argon, and finally 1 μl was injected in the GC-ICP-MS system. Detection was performed at masses 118, 119 and 120. The concentration of the butyltin compounds in the samples was calculated based on the average value from both the 118/119 ratio and the 120/119 ratio. In this way mass bias was compensated without resorting to external mass bias correction. Instrumental detection limits were ca. 50 fg for all butyltin species (as Sn) while method detection limits were blank limited and resulted to be 0.27, 0.09 and 0.04 ng l−1 (as Sn) for TBT, DBT and MBT, respectively. Recovery experiments both at 10 and 70 ng l−1 levels for TBT were quantitative and precision was about 5% RSD at levels between 2 and 20 ng l−1 for all three butyltin compounds. The method was applied to investigate the levels of butyltin compounds in coastal sea-water from Gijon (Asturias, Spain) where no previous data existed.

Development of a triple spike methodology for validation of butyltin compounds speciation analysis by isotope dilution mass spectrometry. Part I. Synthesis of the spike, characterisation and development of the mathematical equations

A methodology for the determination of butyltin compounds by isotope dilution mass spectrometry based on a triple spike approach is presented. A spike solution containing monobutyltin, dibutyltin and tributyltin, each compound enriched with different tin isotopes, has been synthesised, characterised and applied to the simultaneous determination of the three butyltin compounds by isotope dilution GC-ICP-MS. Mathematical equations correcting for all possible degradation reactions which could occur during sample pre-treatment were developed, allowing the accurate calculation of the three butyltin concentrations sought plus the values of six possible interconversion yieids, regardless of the extent of degradation. The triple tin spike solution has shown itself to be stable over more than 7 months, both in isotope composition and elemental concentration, provided that acetic acid is present in the solution. The proposed methodology has been finally evaluated by performing the determination of butyltin compounds in the certified reference material CRM-477 (Mussel Tissue), under different microwave assisted extraction conditions, with satisfactory results.

HPLC-ICPMS and Stable Isotope-Labeled Approaches To Assess Quantitatively Ti(IV) Uptake by Transferrin in Human Blood Serum

Little is known about the effects of titanium found in patients wearing prostheses or about the biochemical pathways of this metal when used as an anticancer drug (e.g., titanocene dichloride). In this work, transferrin has been confirmed as the only carrier protein binding Ti in human blood serum samples by making use of different HPLC protein separations followed by element-specific Ti detection by ICPMS. Besides, isotope dilution analysis has been applied to the quantitative speciation of Ti-Tf in standards and human blood serum samples. Species-unspecific and species-specific isotope dilution modes have been explored. In the first case, very low Ti-Tf results were obtained even using two different chromatographic mechanisms, anion exchange (20-24%) and size exclusion (33-36%). Surprisingly, no major Ti species except Ti-Tf were observed in the chromatograms, suggesting that Ti(IV) hydrolysis and precipitation as inactive titanium oxide species could take place inside the chromatographic columns. These results demonstrate that chemical degradation of metalloproteins during analytical separations could ruin the sought speciation quantitative results. The isotope dilution species-specific mode, much more accurate in such cases, has been instrumental in demonstrating the possibility of gross errors in final metalloprotein quantification. For this purpose, an isotopically enriched standard of (49)Ti-Tf was synthesized and applied to the quantitative speciation of Ti-Tf again. Using this species-specific spike, Ti-Tf dissociation inside the chromatographic columns used could be corrected, and thus, quantitative Ti-Tf binding in serum (92-102%) was observed. In other words, the usefulness and potential of a species-specific isotope dilution analysis approach to investigate quantitatively metal-protein associations, which can be dissociated at certain experimental conditions, is demonstrated here for the first time.

Mass spectrometry for the characterisation of nanoparticles.

Mass spectrometry (MS) has gained much importance in recent years as a powerful tool for reliable analytical characterisation of nanoparticles (NPs). The outstanding capabilities of different MS-based techniques including elemental and molecular detection and their coupling with different separation techniques and mechanisms are outlined herein. Examples of highly valuable elemental and molecular information for a more complete characterisation of NPs are given. Some selected applications illustrate the analytical potential of MS for NP sizing and quantitative assessment of the size distribution as well.