Enhanced Universal Quantification of Biomolecules Using Element MS and Generic Standards: Application to Intact Protein and Phosphoprotein Determination.

Tools that provide absolute quantification of biomolecules, particularly of proteins and their post-translational modifications, without needing suitable specific standards, are urgently demanded nowadays. To this end, we have significantly improved the recently introduced strategy based on CH4 addition to the plasma for absolute quantification of biomolecules using HPLC-ICP-MS. Addition of CO2 has been optimized and finally selected as a safer, more efficient quantitative strategy that is able to provide constant (<6% error) signal response factor for the six elements assayed (S, P, As, Se, Br, I) under compromised conditions. In the particular case of absolute protein quantification, accuracy and precision attainable for S-based absolute determination of intact proteins using internal and external S-generic standards were compared. Potential for real sample analysis was demonstrated by the high-sensitivity analysis of toxins present in snake venoms. Finally, multielemental speciation capabilities of the approach have been also demonstrated through P and S simultaneous analysis in phosphoproteomics. Simultaneous accurate determination of both absolute protein amount and corresponding phosphorylation degree for intact ß-casein, and even impurity traces of κ and α-s1 isoforms present, has been successfully achieved using a simple mixture of inorganic P and S standards. The lowest detection limits (<1 fmol protein) ever published for S- and P-based intact protein quantification with ICP-MS are reported.

Standardization approaches in absolute quantitative proteomics with mass spectrometry.

Mass spectrometry-based approaches have enabled important breakthroughs in quantitative proteomics in the last decades. This development is reflected in the better quantitative assessment of protein levels as well as to understand post-translational modifications and protein complexes and networks. Nowadays, the focus of quantitative proteomics shifted from the relative determination of proteins (ie, differential expression between two or more cellular states) to absolute quantity determination, required for a more-thorough characterization of biological models and comprehension of the proteome dynamism, as well as for the search and validation of novel protein biomarkers. However, the physico-chemical environment of the analyte species affects strongly the ionization efficiency in most mass spectrometry (MS) types, which thereby require the use of specially designed standardization approaches to provide absolute quantifications. Most common of such approaches nowadays include (i) the use of stable isotope-labeled peptide standards, isotopologues to the target proteotypic peptides expected after tryptic digestion of the target protein; (ii) use of stable isotope-labeled protein standards to compensate for sample preparation, sample loss, and proteolysis steps; (iii) isobaric reagents, which after fragmentation in the MS/MS analysis provide a final detectable mass shift, can be used to tag both analyte and standard samples; (iv) label-free approaches in which the absolute quantitative data are not obtained through the use of any kind of labeling, but from computational normalization of the raw data and adequate standards; (v) elemental mass spectrometry-based workflows able to provide directly absolute quantification of peptides/proteins that contain an ICP-detectable element. A critical insight from the Analytical Chemistry perspective of the different standardization approaches and their combinations used so far for absolute quantitative MS-based (molecular and elemental) proteomics is provided in this review.

MMP-11 as a biomarker for metastatic breast cancer by immunohistochemical-assisted imaging mass spectrometry.

MMP-11 is a member of the matrix metalloproteinase family (MMPs) which are overexpressed in cancer cells, stromal cells and the adjacent microenvironment. The MMP protein family encompasses zinc-dependent endopeptidases that degrade the extracellular matrix (ECM), facilitating the breakdown of the basal membrane and matrix connective tissues. This function is believed to be important in cancer development and metastasis. This paper investigated a gold nanoparticle-based immunohistochemical assay to visualise the distribution of MMP-11 in human breast cancer tissues from eight patients with and without metastases by employing laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The expression of MMP-11 was increased and more heterogeneous in metastatic specimens compared to non-metastatic tumour samples. These findings demonstrate that imaging breast tumours by LA-ICP-MS may be a useful tool to aid the prognosis and treatment of breast cancer. As an example, samples of two patients are presented who were diagnosed with matching characteristics and grades of breast cancer. Although both patients had a similar prognosis and treatment, only one developed metastases.

In vivo study of the effect of lactoferrin on iron metabolism and bioavailability from different iron chemical species for formula milk fortification.

Iron fortification in infant formulas is a common practice for providing iron to newborns in order to avoid its deficiency (anemia). Depending on the physicochemical species used, its bioavailability might be insufficient to meet iron requirements. In this vein, the influence of Lactoferrin (Lf) presence on iron bioavailability in 2-week-old wistar rats fed with formula milk fortified with 57 Fe(III)2 -Lf or 57 Fe(II)SO4 (in presence of Lf) using quantitative speciation (by HPLC-ICP-MS) and Isotope Pattern Deconvolution (IPD) is studied here. Results obtained were compared among fortifiers and also with the maternal group. In RBCs, iron was mainly bound to hemoglobin in all the assayed groups in the same extent. Regarding serum samples, several iron-proteins were observed (such as transferrin and albumin). In both samples, iron content in the fractions studied was similar in all groups compared and exogenous 57 Fe incorporation of intaked iron was always above 50%, showing no significative differences between physicochemical forms but related to the dose administered. Regarding iron stores (liver) the group fed with formula milk fortified with the higher dose of 57 FeSO4 in presence of Lf presented the highest values of total iron even superior than those found in the maternal group, and also the highest exogenous (57 Fe) incorporation. In conclusion, it was proved that iron fortification is required to ensure proper iron levels in all body compartments. No significative differences were observed between different physicochemical species when iron is administered at low doses. However, higher iron doses lead to a greater incorporation in all the iron-proteins studied.

Capping of Mn-Doped ZnS Quantum Dots with DHLA for Their Stabilization in Aqueous Media: Determination of the Nanoparticle Number Concentration and Surface Ligand Density.

Colloidal Mn2+-doped ZnS quantum dots (QDs) were synthesized, surface modified, and thoroughly characterized using a pool of complementary techniques. Cap exchange of the native l-cysteine coating of the QDs with dihydrolipoic acid (DHLA) ligands is proposed as a strategy to produce nanocrystals with a strong phosphorescent-type emission and improved aqueous stability. Moreover, such a stable DHLA coating can facilitate further bioconjugation of these QDs to biomolecules using established reagents such as cross-linker molecules. First, a structural and morphological characterization of the l-cysteine QD core was performed by resorting to complementary techniques, including X-ray powder diffraction (XRD) and microscopy tools. XRD patterns provided information about the local structure of ions within the nanocrystal structure and the number of metal atoms constituting the core of a QD. The judicious combination of the data obtained from these complementary characterization tools with the analysis of the QDs using inductively coupled plasma-mass spectrometry (ICP-MS) allowed us to assess the number concentration of nanoparticles in an aqueous sample, a key parameter when such materials are going to be used in bioanalytical or toxicological studies. Asymmetric flow field-flow fractionation (AF4) coupled online to ICP-MS detection proved to be an invaluable tool to compute the number of DHLA molecules attached to the surface of a single QD, a key feature that is difficult to estimate in nanoparticles and that critically affects the behavior of nanoparticles when entering the biological media (e.g., cellular uptake, biodistribution, or protein corona formation). This hybrid technique also allowed us to demonstrate that the elemental composition of the nanoparticle core remains unaffected after the ligand exchange process. Finally, the photostability and robustness of the DHLA-capped QDs, critical parameters for bioanalytical applications, were assessed by molecular luminescence spectroscopy.

Iron bioavailability from supplemented formula milk: effect of lactoferrin addition.

PURPOSE: In this work, the absorption and/or bioavailability of iron from two chemical species, 57Fe-Lf (apo-lactoferrin) complex and 57FeSO4 at low and high dose, and in Lf excess were investigated in lactating wistar rats. METHODS: The methodology used is based on the use of stable isotopes in combination with the approach "isotope pattern deconvolution" and ICP-MS for detection. This approach provides quantitative information about exogenous (57Fe) and endogenous iron (natFe) distribution in fluids and tissues in the iron-supplemented rat groups. RESULTS: The observed results with supplemented rats were compared with those found in rats receiving maternal feeding. Interestingly, differences were found between groups in iron for transport and storage compartments, but not in the functional one, depending upon the dose of iron administered and the chemical species. CONCLUSION: Considering the results obtained, supplementation with iron salts in excess of Lf appears to be the best way of iron supplementation of formula milk.

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.

A flowing atmospheric pressure afterglow as an ion source coupled to a differential mobility analyzer for volatile organic compound detection.

Atmospheric pressure glow discharges have been widely used in the last decade as ion sources in ambient mass spectrometry analyses. Here, an in-house flowing atmospheric pressure afterglow (FAPA) has been developed as an alternative ion source for differential mobility analysis (DMA). The discharge source parameters (inter-electrode distance, current and helium flow rate) determining the atmospheric plasma characteristics have been optimized in terms of DMA spectral simplicity with the highest achievable sensitivity while keeping an adequate plasma stability and so the FAPA working conditions finally selected were: 35 mA, 1 L min(-1) of He and an inter-electrode distance of 8 mm. Room temperature in the DMA proved to be adequate for the coupling and chemical analysis with the FAPA source. Positive and negative ions for different volatile organic compounds were tested and analysed by FAPA-DMA using a Faraday cup as a detector and proper operation in both modes was possible (without changes in FAPA operational parameters). The FAPA ionization source showed simpler ion mobility spectra with narrower peaks and a better, or similar, sensitivity than conventional UV-photoionization for DMA analysis in positive mode. Particularly, the negative mode proved to be a promising field of further research for the FAPA ion source coupled to ion mobility, clearly competitive with other more conventional plasmas such as corona discharge.

Aqueous synthesis of near-infrared highly fluorescent platinum nanoclusters.

A one-step synthesis of near infrared fluorescent platinum nanoclusters (PtNCs) in aqueous medium is described. The proposed optimized procedure for PtNC synthesis is rather simple, fast and it is based on the direct metal reduction with NaBH4. Bidentated thiol ligands (lipoic acid) were selected as nanoclusters stabilizers in water media. The structural characterization revealed attractive features of the PtNCs, including small size, high water solubility, near-infrared luminescence centered at 680 nm, long-term stability and the highest quantum yield in water reported so far (47%) for PtNCs. Moreover, their stability in different pH media and an ionic strength of 0.2 M NaCl was studied and no significant changes in fluorescence emission were detected. In brief, they offer a new type of fluorescent noble metal nanoprobe with a great potential to be applied in several fields, including biolabeling and imaging experiments.

Determination of reduced homocysteine in human serum by elemental labelling and liquid chromatography with ICP-MS and ESI-MS detection.

Analytical methods allowing sensitive determination of reduced homocysteine (rHcy), one of the so-called biothiols, in human serum is a topic of growing interest due to its close relation to several human disorders, mainly cardiovascular diseases. Although most widely used analytical strategies to determine total Hcy involve derivatization by means of fluorescent labels, this work proposes the use of ebselen, a Se-containing labelling agent to derivatize the reactive sulfhydryl group of the Hcy molecule in its "free" reduced form, which is more likely to play different roles in disease pathogenesis. Optimization of the derivatization and separation conditions by high-performance liquid chromatography (HPLC) to isolate the excess of derivatizing reagent is carried out here using UV/VIS detection. Further, the study of the Se labelling reaction by electrospray ionization tandem mass spectrometry (ESI-MS/MS) provides a stoichiometry of the derivative of 1:1. The main advantage of using ebselen as a labelling agent is the presence of the Se atom in the molecule that allows the use of inductively coupled plasma mass spectrometry (ICP-MS) as a sensitive and selective Se detector. The coupling of HPLC with ICP-MS provided excellent features for the determination of Se-derivatized rHcy (detection limit of 9.6 nM) in real samples. Quantification was accomplished by using post-column isotope dilution (ID) of Se in serum samples, after precipitation of the main serum proteins. Quantitative results for "free" rHcy turned out to be around 0.18-0.22 µM in serum samples from healthy individuals that could be directly analyzed without sample preconcentration.

Quantitative selenium speciation by HPLC-ICP-MS(IDA) and simultaneous activity measurements in human vitreous humor.

A post-column isotope dilution analysis (IDA) methodology was applied to carry out quantitative speciation of selenium in human vitreous humor samples by size exclusion chromatography (SEC) coupled on-line to inductively coupled plasma mass spectrometry (ICP-MS). Two main selenium species detected by SEC-ICP-MS were found to be associated to protein complexes. The expected molecular weights for both selenium-bound complexes were confirmed by MALDI-TOF(MS) and the results matched well with the theoretical mass of a GPx monomer (M, 22 kDa) and tetramer (T, 88 kDa). The quantification of the two detected selenium-bound complexes by post-column IDA showed that the total content of selenospecies in vitreous humor was approximately 3.2 ± 1.8 ppb Se. Moreover, in most of the analyzed vitreous humor samples, the majority of the selenium was associated to higher molecular weight GPx biomolecules. In an attempt to assess if the enzymatic activity was associated with a given selenium-bound GPx protein, the antioxidant enzyme activity was assayed for the two separated GPx species. Only for GPx (T) was a linear relationship between activity and total Se concentration found by ICP-MS.

Improving pulsed radiofrequency glow discharge for time-of-flight mass spectrometry simultaneous elemental and molecular analysis.

The performance of radiofrequency (rf) millisecond pulsed glow discharge (PGD) coupled to a fast orthogonal time-of-flight mass spectrometer (TOFMS) for chemical characterization and quantification of organic volatile compounds was investigated by using two different GD chamber designs. The designs investigated had substantial differences in the way that the volatile organic compound is introduced into the GD and the distance between the cathode and the sampling cone of the mass spectrometer. Bromochloromethane was selected as the model analyte because of the practical interest of determining trihalomethanes at low concentrations, and also because of both its low boiling point (to avoid problems associated with condensations in the interface) and the fact that it has two different heteroatoms, making the fragmentation patterns easier to follow. Pulse shapes of element, fragment, and molecular parent ions obtained by using the two GD chambers under investigation were critically compared. Results revealed the critical effect of the GD chamber geometry in obtaining the three types of chemical information, temporally discriminated. The spectra of the gaseous samples and of a polymer containing TBBPA (solid sample) were also compared. Detection limits for bromochloromethane in the order of low ng L(-1), and the required high tolerance of the plasmas to the introduction of organic vapours, were achieved using one of the proposed GD designs. The capability of the designed system for the analysis of other volatile compounds, for example dimethyl disulfide and dimethyl selenide, was also successfully evaluated, making use of the analytical potential of the information obtained from the different pulse time regions.

Effect of internal and external conditions on ionization processes in the FAPA ambient desorption/ionization source.

Ambient desorption/ionization (ADI) sources coupled to mass spectrometry (MS) offer outstanding analytical features: direct analysis of real samples without sample pretreatment, combined with the selectivity and sensitivity of MS. Since ADI sources typically work in the open atmosphere, ambient conditions can affect the desorption and ionization processes. Here, the effects of internal source parameters and ambient humidity on the ionization processes of the flowing atmospheric pressure afterglow (FAPA) source are investigated. The interaction of reagent ions with a range of analytes is studied in terms of sensitivity and based upon the processes that occur in the ionization reactions. The results show that internal parameters which lead to higher gas temperatures afforded higher sensitivities, although fragmentation is also affected. In the case of humidity, only extremely dry conditions led to higher sensitivities, while fragmentation remained unaffected.

Quantitative bioimaging of trace elements in the human lens by LA-ICP-MS.

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used for the quantitative imaging of Fe, Cu and Zn in cryostat sections of human eye lenses and for depth profiling analysis in bovine lenses. To ensure a tight temperature control throughout the experiments, a new Peltier-cooled laser ablation cell was employed. For quantification purposes, matrix-matched laboratory standards were prepared from a pool of human lenses from eye donors and spiked with standard solutions containing different concentrations of natural abundance Fe, Cu and Zn. A normalisation strategy was also carried out to correct matrix effects, lack of tissue homogeneity and/or instrumental drifts using a thin gold film deposited on the sample surface. Quantitative images of cryo-sections of human eye lenses analysed by LA-ICP-MS revealed a homogeneous distribution of Fe, Cu and Zn in the nuclear region and a slight increase in Fe concentration in the outer cell layer (i.e. lens epithelium) at the anterior pole. These results were assessed also by isotope dilution mass spectrometry, and Fe, Cu and Zn concentrations determined by ID-ICP-MS in digested samples of lenses and lens capsules.

Changes in iron metabolism and oxidative status in STZ-induced diabetic rats treated with bis(maltolato) oxovanadium (IV) as an antidiabetic agent.

The role of vanadium as a micronutrient and hypoglycaemic agent has yet to be fully clarified. The present study was undertaken to investigate changes in the metabolism of iron and in antioxidant defences of diabetic STZ rats following treatment with vanadium. Four groups were examined: control; diabetic; diabetic treated with 1 mgV/day; and Diabetic treated with 3 mgV/day. The vanadium was supplied in drinking water as bis(maltolato) oxovanadium (IV) (BMOV). The experiment had a duration of five weeks. Iron was measured in food, faeces, urine, serum, muscle, kidney, liver, spleen, and femur. Superoxide dismutase, catalase, NAD(P)H: quinone-oxidoreductase-1 (NQO1) activity, and protein carbonyl group levels in the liver were determined. In the diabetic rats, higher levels of Fe absorbed, Fe content in kidney, muscle, and femur, and NQO1 activity were recorded, together with decreased catalase activity, in comparison with the control rats. In the rats treated with 3 mgV/day, there was a significant decrease in fasting glycaemia, Fe content in the liver, spleen, and heart, catalase activity, and levels of protein carbonyl groups in comparison with the diabetic group. In conclusion BMOV was a dose-dependent hypoglycaemic agent. Treatment with 3 mgV/day provoked increased Fe deposits in the tissues, which promoted a protein oxidative damage in the liver.

The stoichiometric transition from Zn6Cu1-metallothionein to Zn7-metallothionein underlies the up-regulation of metallothionein (MT) expression: quantitative analysis of MT-metal load in eye cells.

We examined the profiling of gene expression of metallothioneins (MTs) in human tissues from cadaver eyes with microarray-based analysis. All MT1 isoforms, with the exception of MT1B, were abundantly expressed in lens and corneal tissue. Along with MT1B, MT4 was not detected in any tissues. Antibodies to MT1/2 labeled the corneal epithelial and endothelial cells, whereas MT3 label the retinal ganglion cells. We studied the effects of zinc and cytokines on the gene expression of MT isoforms in a corneal epithelial cell line (HCEsv). Zinc exerted an up-regulation of the expression of MT isoforms, and this effect was further potentiated in the presence of IL1α or TNFα. Zinc also elicited a strong down-regulation of the expression of inflammatory cytokines, and this effect was blocked in the presence of TNFα or IL1α. The concentration of MTs, bound zinc, and the metal stoichiometry of MTs in cultured HCEsv were determined by mass spectrometry. The total concentration of MTs was 0.24 ± 0.03 µM and, after 24 h of zinc exposure, increased to 0.96 ± 0.01 µM. The combination of zinc and IL1α further enhanced the level of MTs to 1.13 ± 0.03 µM. The average metal stoichiometry of MTs was Zn(6)Cu(1)-MT, and after exposure to the different treatments, it changed to Zn(7)-MT. Actinomycin D blocked transcription, and cycloheximide attenuated synthesis of MTs in the presence or absence of zinc, suggesting transcriptional regulation. Overall the data provide molecular and analytical evidence on the interplay between zinc, MTs, and proinflammatory cytokines in HCEsv cells, with potential implications on cell-based inflammatory eye diseases.

Liquid chromatography, chemical oxidation, and online carbon isotope dilution mass spectrometry as a universal quantification system for nonvolatile organic compounds.

A procedure for the universal detection and quantification of polar organic compounds separated by liquid chromatography (LC) based on postcolumn carbon isotope dilution mass spectrometry (IDMS) was developed. The eluent from the LC column is mixed online with a continuous flow of (13)C-enriched sodium bicarbonate, and the sodium persulfate oxidation reaction in acidic media is employed to achieve isotope equilibration. All carbon-containing compounds eluting from the column are oxidized to (12)CO(2) and (13)CO(2), respectively, and the carbon dioxide is separated from the aqueous phase using a gas-permeable membrane. The gaseous carbon dioxide is then carried to the mass spectrometer for isotope ratio measurements. Different water-soluble organic compounds were evaluated using a flow injection configuration to assess the efficiency of the oxidation process. Most water-soluble organic compounds tested showed quantitative oxidation. However, chemical structures involving conjugated C═N double bounds and guanidinium-like structures were found to be resistant to the oxidation and were further studied. For this purpose, (13)C(1)-labeled creatine (with the isotopic label in the guanidinium group) was employed as model compound. Specific conditions for the quantitative oxidation of these compounds required lower flow rates and the addition of metallic catalysts. This novel approach was tested as a universal detection and quantification system for LC. A simple standard mixture of four amino acids was separated under 100% aqueous conditions and quantified without the need for specific standards with good accuracy and precision using potassium hydrogen phthalate as internal standard. The main field of application of the developed method is for the purity assessment of organic standards with direct traceability to the International System of Units (SI).

One-step aqueous synthesis of fluorescent copper nanoclusters by direct metal reduction.

A one-step aqueous synthesis of highly fluorescent water-soluble copper nanoclusters (CuNCs) is here described, based on direct reduction of the metal precursor with NaBH4 in the presence of bidentate ligands (made of lipoic acid anchoring groups, appended with a poly(ethylene glycol) short chain). A complete optical and structural characterization was carried out: the optical emission was centred at 416 nm, with a luminescence quantum yield in water of 3.6% (the highest one reported so far in water for this kind of nanocluster). The structural characterization reveals a homogeneous size distribution (of 2.5 nm diameter) with spherical shape. The CuNCs obtained offer long-term stability (the luminescence emission remained unaltered after more than two months) under a broad range of chemical conditions (e.g., stored at pH 3-12 or even in a high ionic strength medium such as 1 M NaCl) and high photostability, keeping their fluorescence emission intact after more than 2 h of daylight and UV-light exposition. All those advantageous features warrant synthesized CuNCs being promising fluorescent nanoprobes for further developments including (bio)applications.

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.

Critical evaluation of the potential of radiofrequency pulsed glow discharge-time-of-flight mass spectrometry for depth-profile analysis of innovative materials.

The combination of radiofrequency pulsed glow discharge (RF-PGD) analytical plasmas with time-of-flight mass spectrometry (TOFMS) has promoted the applicability of this ion source to direct analysis of innovative materials. In this sense, this emerging technique enables multi-elemental depth profiling with high depth resolution and sensitivity, and simultaneous production of elemental, structural, and molecular information. The analytical potential and trends of this technique are critically presented, including comparison with other complementary and well-established techniques (e.g. SIMS, GD-OES, etc.). An overview of recent applications of RF-PGD-TOFMS is given, including analysis of nano-structured materials, coated-glasses, photovoltaic materials, and polymer coatings.