Total metal content and chemical speciation analysis of iron, copper, zinc and iodine in human breast milk using high-performance liquid chromatography separation and inductively coupled plasma mass spectrometry detection.

The aim of this research was to quantify essential trace elements (iron, copper, zinc and iodine) and establish their speciation in human milk. Both the element and the species are important in new-born nutrition. Colostrum, and transitional and mature milks (25) were collected from 18 mothers of pre-term or full-term infants. Concentrations of the target elements were determined using ICP-MS. For speciation, HPLC coupled to ICP-MS was employed. Total contents of the micronutrients varied in mothers of pre-term (Fe = 0.997, Cu = 0.506, Zn = 4.15 and I = 0.458 mg L-1) and mothers of full-term (Fe = 0.733, Cu = 0.234, Zn = 2.91 and I = 0.255 mg L-1) infants. Fe, Cu and Zn were associated with biomolecules with high molecular mass compounds, such as immunoglobulins, albumin and lactoferrin whilst iodine was only found as iodide.

Capabilities of asymmetrical flow field - Flow fractionation on-line coupled to different detectors for characterization of water-stabilized quantum dots bioconjugated to biomolecules.

The asymmetric flow field-flow fractionation (AF4) coupled on-line with elemental (inductively coupled plasma-mass spectrometry, ICP-MS) and molecular (fluorescence and UV) detection has been investigated as a powerful tool for the characterization of bioinorganic nano-conjugates. In this study, we described methods for the characterization of biotin-antibody complexes bioconjugated with streptavidin quantum dots (QDs-SA-b-Ab). Operating parameters of AF4 separation technique were optimized and two procedures are proposed using a channel thickness of 350 µm and 500 µm. The use of a 500 µm spacer allowed to achieve an efficient AF4 separation of the QDs-SA-b-Ab complexes from the excess of individual species used in the bioconjugation that was required for a proper characterization of the bioconjugates. Optimization of the AF4 allowed a separation resolution good enough to isolate the QDs-SA-b-Ab bioconjugates from the free excess of b-Ab and QD-SA. The efficiency of the bioconjugation process could be then calculated, obtaining a value of 86% for a 1 QDs-SA: 5 b-Ab bioconjugation ratio. In addition, sample recovery around 90% was achieved.

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.

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.

Universal absolute quantification of biomolecules using element mass spectrometry and generic standards.

We present a novel and highly sensitive ICP-MS approach for absolute quantification of all important target biomolecule containing P, S, Se, As, Br, and/or I (e.g., proteins and phosphoproteins, metabolites, pesticides, drugs), under the same simple instrumental conditions and without requiring any specific and/or isotopically enriched standard.

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.

Protective effect of selenium supplementation following oxidative stress mediated by glucose on retinal pigment epithelium.

There are many conditions that affect the retina. However, diabetic retinopathy (RD) as a complication of Diabetes Mellitus continues to be the leading cause of blindness in working people globally. Diabetic retinopathy is an ocular complication of diabetes that is caused by the deterioration of the blood vessels that supply the retina, which has the consequence that the vision deteriorates irreversibly. The retina, and specifically the retinal pigment epithelium (RPE) is the only neural tissue that is exposed directly and frequently to light, which favors the oxidation of lipids that become extremely toxic to the cells of the retina. The RPE is a natural barrier playing an important role in the absorption of light and reduction of light scatter within the eye. In addition, the retina is the tissue that proportionally consumes more oxygen, which generates a high production of reactive oxygen species (ROS). The retina is particularly sensitive to hyperglycemia and oxidative stress. The eye tissues are enriched in certain antioxidants in the form of metabolic enzymes or small molecules. Since selenium is essential for regulating the activity of the enzymes involved in protection against oxidative stress, providing selenium to the ocular tissues could be useful for the treatment of different ocular pathologies. Thus, the aim of this study is to investigate the potential efficacy of selenium in human RPE against glucose-induced oxidative stress and its implications for GPx activity. Chromatographic techniques based on HPLC-ICP-MS will be applied in combination with isotope pattern deconvolution (IPD) to study the effects of selenium supplementation and hyperglycemia in an in vitro model of RPE cells.

Quantitative study of zinc and metallothioneins in the human retina and RPE cells by mass spectrometry-based methodologies.

The retina contains the highest concentration of zinc in the human eye and it is primarily associated with the retinal pigment epithelium (RPE). Metallothioneins (MTs) are the main cytosolic zinc-ion-binding proteins, exerting a tight control in the number of atoms of Zn-bound to the MTs related with their antioxidant and neuroprotective functions. In order to study the Zn-MT system in retina and RPE, we have implemented mass spectrometry (MS)-based technologies: two complementary element detection methodologies (HPLC- and laser ablation (LA)-ICP-MS) have been successfully employed to study metal content in the human eye as well as to perform speciation studies of Zn-MTs. First, Zn-elemental distribution was studied on cryogenic ocular sections by LA-ICP-MS. Quantitative images of Zn along RPE cell layer and the retina were obtained with a laser beam diameter of 25µm, showing a preferential distribution in the RPE. We carried out then the quantitative speciation of Zn, Fe, and Cu in the water-soluble protein fractions of RPE and retina to study their protein binding profile using HPLC-ICP-MS, where Zn is mainly associated to low molecular mass proteins (i.e., MTs). Finally, the effect of addition of different inductors, such as metal (i.e., 68ZnSO4), dexamethasone (DEX) and erythropoietin, was investigated in an in vitro cellular model of human RPE cells (HRPEsv), again using HPLC-ICP-MS in combination with stable isotopes and mathematical calculations based on isotope dilution and isotope pattern deconvolution. Exogenous Zn and DEX were found to increase MT proteins synthesis and exerted a stoichiometric transition in MT proteins in HRPEsv cells.

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.

Sensitive prostate specific antigen quantification using dihydrolipoic acid surface-functionalized phosphorescent quantum dots.

Herein, high-quality Mn-doped ZnS quantum dots (QDs) have been synthesized using a facile approach directly in aqueous media. The surface of the obtained QDs was further modified by cap-exchange of the native cysteine shell with dihydrolipoic acid (DHLA) ligands resulting in nanocrystals with high water-stability having an intense phosphorescent signal. Covalent bioconjugation of the DHLA-coated nanoparticles with an anti-IgG antibody was then carried out. Interestingly the QD immunoprobe (QD-labelled antibodies) maintained an intense phosphorescence emission, without any significant spectral-shift (as compared to the free QDs). Coupling of an asymmetric flow field flow fractionation technique to an elemental mass spectrometry detection enabled the accurate determination of the efficiency of the bioconjugation reaction. The obtained nanoparticle-antibody bioconjugate was then applied to develop a quantitative sandwich-type phosphorescent immunoassay for Prostate Specific Antigen (PSA), and a limit of detection (LOD) of 17 pg mL-1 of PSA was achieved and allow to quantify such biomarker in samples within clinically relevant levels. Finally, the assay was validated for the quantification of PSA in the cellular media of prostate cancer cells. Obtained results proved the robustness of the proposed immunoassay based on long-lived phosphorescence measurements against eventual photoluminescent interferences significantly affecting the conventional short-lived fluorescence detection.

Assessment of the removal of side nanoparticulated populations generated during one-pot synthesis by asymmetric flow field-flow fractionation coupled to elemental mass spectrometry.

Coupling of asymmetric flow field-flow fractionation (AF4) to an on-line elemental detection (inductively coupled plasma-mass spectrometry, ICP-MS) has been recently proposed as a powerful diagnostic tool for characterization of the bioconjugation of CdSe/ZnS core-shell Quantum Dots (QDs) to antibodies. Such approach has been used herein to demonstrate that cap exchange of the native hydrophobic shell of core/shell QDs with the bidentate dihydrolipoic acid ligands directly removes completely the eventual side nanoparticulated populations generated during simple one-pot synthesis, which can ruin the subsequent final bioapplication. The critical assessment of the chemical and physical purity of the surface-modified QDs achieved allows to explain the transmission electron microscopy findings obtained for the different nanoparticle surface modification assayed.

Absolute venomics: Absolute quantification of intact venom proteins through elemental mass spectrometry.

We report the application of a hybrid element and molecular MS configuration for the parallel absolute quantification of µHPLC-separated intact sulfur-containing venom proteins, via ICP triple quadrupole MS and 32S/34S isotope dilution analysis, and identification by ESI-QToF-MS of the toxins of the medically important African black-necked spitting cobra, Naja nigricollis (Tanzania); New Guinea small-eyed snake, Micropechis ikaheka; and Papuan black snake, Pseudechis papuanus. The main advantage of this approach is that only one generic sulfur-containing standard is required to quantify each and all intact Cys- and/or Met-containing toxins of the venom proteome. The results of absolute quantification are in reasonably good agreement with previously reported relative quantification of the most abundant protein families. However, both datasets depart in the quantification of the minor ones, showing a tendency for this set of proteins to be underestimated in standard peptide-centric venomics approaches. The molecular identity, specific toxic activity, and concentration in the venom, are the pillars on which the toxicovenomics-aimed discovery of the most medically-relevant venom toxins, e.g. those that need to be neutralized by an effective therapeutic antivenom, should be based. The pioneering venom proteome-wide absolute quantification shown in this paper represents thus a significant advance towards this goal. The potential of ICP triple quadrupole MS in proteomics in general, and venomics in particular, is critically discussed. BIOLOGICAL SIGNIFICANCE: Animal venoms provide excellent model systems for investigating interactions between predators and prey, and the molecular mechanisms that contribute to adaptive protein evolution. On the other hand, numerous cases of snake bites occur yearly by encounters of humans and snakes in their shared natural environment. Snakebite envenoming is a serious global public health issue that affects the most impoverished and geopolitically disadvantaged rural communities in many tropical and subtropical countries. Unveiling the temporal and spatial patterns of venom variability is of fundamental importance to understand the molecular basis of envenoming, a prerequisite for developing therapeutic strategies against snakebite envenoming. Research on venoms has been continuously enhanced by advances in technology. The combined application of next-generation transcriptomic and venomic workflows has demonstrated unparalleled capabilities for venom characterization in unprecedented detail. However, mass spectrometry is not inherently quantitative, and this analytical limitation has sparked the development of methods to determine absolute abundance of proteins in biological samples. Here we show the potential of a hybrid element and molecular MS configuration for the parallel ESI-QToF-MS and ICP-QQQ detection and absolute quantification of intact sulfur-containing venom proteins via 32S/34S isotope dilution analysis. This configuration has been applied to quantify the toxins of the medically important African snake Naja nigricollis (Tanzania), and the Papuan species Micropechis ikaheka and Pseudechis papuanus.

Volatile organic compound analysis by pulsed glow discharge time of flight mass spectrometry as a structural elucidation tool.

Pulsed glow discharge (PGD) coupled to time of flight mass spectrometry (TOFMS) has been investigated for volatile organic compound (VOC) identification and determination. Optimization of PGD operational conditions (chamber design, applied power, pressure and duty cycle) was performed using acetone and benzene as model compounds. During the different optimizations, molecular, fragment and elemental information were obtained when characteristic GD pulse regions were measured. An exploratory study for several VOCs (lineal hydrocarbons, oxygen-containing compounds and aromatic compounds) revealed the capability of the PGD to provide crucial information to elucidate structures (fragments), molecular ions or even proton affinity nature of the molecules; this last information is a consequence of the enriched proton environment generated along the afterglow region for the ionization chamber used. Analytical characteristics were evaluated with solid phase microextraction-gas chromatography coupled to PGD-TOFMS for special aromatic hydrocarbons (benzene, toluene, ethylbenzene, xylene: BTEX) in water, showing a good performance in terms of reproducibility and sensitivity. Copyright © 2017 John Wiley & Sons, Ltd.

Protein-species quantitative venomics: looking through a crystal ball.

In this paper we discuss recent significant developments in the field of venom research, specifically the emergence of top-down proteomic applications that allow achieving compositional resolution at the level of the protein species present in the venom, and the absolute quantification of the venom proteins (the term "protein species" is used here to refer to all the different molecular forms in which a protein can be found. Please consult the special issue of Jornal of Proteomics "Towards deciphering proteomes via the proteoform, protein speciation, moonlighting and protein code concepts" published in 2016, vol. 134, pages 1-202). Challenges remain to be solved in order to achieve a compact and automated platform with which to routinely carry out comprehensive quantitative analysis of all toxins present in a venom. This short essay reflects the authors' view of the immediate future in this direction for the proteomic analysis of venoms, particularly of snakes.

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.

Characterization of thin film tandem solar cells by radiofrequency pulsed glow discharge - Time of flight mass spectrometry.

Beside low production costs and the use of nontoxic and abundant raw materials, silicon based thin-film solar cells have the advantage to be built up as multi junction devices like tandem or triple junction solar cells. Silicon thin film modules made of tandem cells with hydrogenated amorphous silicon (a-Si:H) top cell and microcrystalline (µc) Si:H bottom cell are available on the market. In this work, the analytical potential of state-of-the art radiofrequency (rf) pulsed glow discharge (PGD) time of flight mass spectrometry (TOFMS) commercial instrumentation is investigated for depth profiling analysis of tandem-junctions solar cells on 2mm thick glass substrate with 1µm thick ZnO:Al. Depth profile characterization of two thin film tandem photovoltaic devices was compared using millisecond and sub-millisecond rf-PGD regimes, as well as the so-called "low mass mode" available in the commercial instrument used. Two procedures for sample preparation, namely using flat or rough cell substrates, were compared and the distribution of dopant elements (phosphorous, boron and germanium) was investigated in both cases. Experimental results obtained by rf-PGD-TOFMS as well as electrical measurements of the samples showed that a worse depth resolution of dopant elements in the silicon layers (e.g. distribution of boron in a thicker region that suggests a diffusion of this dopant in the coating of the sample) found using a rough sample substrate was related to a higher power conversion efficiency.

Elemental and isotopic analysis of oral squamous cell carcinoma tissues using sector-field and multi-collector ICP-mass spectrometry.

Elemental and isotopic analysis via single-collector and multi-collector ICP-mass spectrometry, respectively, have been explored as a tool for identifying potential differences between non-tumor and oral squamous cell carcinoma tissues. Elemental concentrations of major and minor elements, known to be essential for different processes in the cell (i.e. Na, Ca, Mg, K, P, Fe, Cu and Zn), have been determined and results for cancerous and non-cancerous tissues collected from the same individual have been compared. Among the elements studied, only Mg, K and P turned out to be significantly higher in concentration in the tumor tissues. However, a shift towards higher and wider concentration ranges has also been observed for Cu and Zn in the tumor samples, whereas for Ca lower concentrations were established. Possible isotope ratio variations for Cu and Zn in both biological tissues have also been evaluated with the same goal. δ66Zn results did not provide an obvious trend, but in the case of Cu, a clear distinction between the tumor and non-tumor tissues was observed: δ65Cu values ranged between -0.68% and 0.03% in the non-tumor tissues, whereas tumor samples turned out to be enriched in 65Cu, with δ65Cu values between 0.10% and 0.93%. These results confirm the considerable potential of isotopic and elemental studies for biomedical purposes.

Effect of vanadium on calcium homeostasis, osteopontin mRNA expression, and bone microarchitecture in diabetic rats.

The aim of this study was to examine whether alterations caused by diabetes in calcium homeostasis, expression of osteopontin and the microarchitecture of bone are corrected by exposure to vanadium. Four study groups were examined over a period of five weeks: control (C), diabetic (DM), diabetic treated with 1 mg V per d (DMV), and diabetic treated with 3 mg V per d (DMVH). Vanadium was supplied in drinking water as bis(maltolato)oxovanadium(iv). Calcium was measured in the food, faeces, urine, serum, kidneys, liver, muscles, and femur. Osteopontin gene expression was determined in the liver, and the bone microarchitecture was studied with the aid of micro-computed tomography. In the DM group, food intake as well as calcium absorbed and retained and liver osteopontin mRNA increased, while Ca in the serum and femur decreased, and the bone microarchitecture worsened, in comparison with the control. In the DMV group, the amount of Ca absorbed and retained was similar to DM rats. Although the Ca content in the femur increased and osteopontin mRNA decreased, there were no significant changes in the bone microarchitecture, in comparison to the DM rats. In the DMVH group, the amount of Ca absorbed and retained, and the serum and femur content were equivalent to the control. The levels of osteopontin mRNA decreased and bone mineralization improved, compared to the DM group. We conclude that treatment with 3 mg V per d of the glucose lowering agent bis(maltolato)oxovanadium(iv) causes a decrease in osteopontin mRNA, which could favour the normalization of changes in Ca homeostasis and bone microarchitecture, both at the cortical and trabecular levels, caused by diabetes.