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The development of methods to analyze accurately and precisely individual metalloproteins is of increasing importance. Here we describe for the first time the chemical preparation and characterization of an isotopically enriched metalloenzyme containing two different metal isotopes. Its evaluation as a standard in species-specific isotope dilution analysis by HPLC coupled to inductively coupled plasma mass spectrometry is carefully evaluated. Our model enzyme bovine superoxide dismutase (SOD) contains both Cu and Zn and is remarkably stable at high temperatures and even under denaturing conditions. The enzyme's metal cofactors were removed under a range of different conditions and replaced with isotopically enriched [sup.65]Cu and [sup.68]Zn. Depending on the conditions, various isotopic ratios differing from the natural Cu and Zn abundances were obtained for the reconstituted enzyme. Both the wild type and isotopicaily enriched enzyme had the same migration pattern on native 1D-PAGE. Using an enzyme activity test, we showed that the incorporated [sup.65]Cu was bound to the right SOD-binding site, since the measured activity correlated directly with the amount of Cu incorporated. Mixing the native and the isotopically enriched enzyme standard with free enriched [sup.65]Cu and [sup.68]Zn or a metal chelator did not result in any exchange or loss of the metals from the enzyme at neutral pH. This verifies the stability of the enzyme metal center under the chosen conditions. The isotopically enriched enzyme standard was spiked into a wild type SOD solution to evaluate its use for species-specific isotope dilution experiments. To our knowledge, this is the first report of the chemical preparation of a metalloenzyme containing two different isotopically enriched metals. We provide evidence that the incorporated isotopically enriched metals are bound to the right binding site of SOD using an specific enzymatic activity assay.

A novel strategy for the absolute quantification of selenium (Se) included in selenoprotein P (SEPP1), an important biomarker for human nutrition and disease, including diabetes and cancer, is presented here for the first time. It is based on the use of species-specific double isotope dilution mass spectrometry (SSIDA) in combination with HPLC-ICP-MS/MS for the determination of protein bound Se down to the peptide level in a complex plasma matrix with a total content of Se of 105.5 µg kg-1. The method enabled the selective Se speciation analysis of human plasma samples without the need of extensive cleanup or preconcentration steps as required for traditional protein mass spectrometric approaches. To assess the method accuracy, two plasma reference materials, namely, BCR-637 and SRM1950, for which literature data and a reference value for SEPP1 have been reported, were analyzed using complementary hyphenated methods and the species-specific approach developed in this work. The Se mass fractions obtained via the isotopic ratios 78Se/76Se and 82Se/76Se for each of the Se-peptides, namely, ENLPSLCSUQGLR (ENL) and AEENITESCQUR (AEE) (where U is SeCys), were found to agree within 2.4%. A relative expanded combined uncertainty (k = 2) of 5.4% was achieved for a Se (as SEPP1) mass fraction of approximately 60 µg kg-1. This work represents a systematic approach to the accurate quantitation of plasma SEPP1 at clinical levels using SSIDA quantification. Such methodology will be invaluable for the certification of reference materials and the provision of reference values to clinical measurements and clinical trials. [ABSTRACT FROM AUTHOR]

The hyphenation of HPLC with its high separation ability and ICP-MS with its excellent sensitivity, allows the analysis of Pt drugs in biological samples at the low nanomolar concentration levels. On the other hand, LC-MS provides molecular structural confirmation for each species. Using a combination of these methods, we have investigated the speciation of the photoactive anticancer complex diazido Pt(IV) complex trans , trans , trans -[Pt(N 3 ) 2 (OH) 2 (py) 2 ] (FM-190) in aqueous solution and biofluids at single-digit nanomolar concentrations before and after irradiation. FM-190 displays high stability in human blood plasma in the dark at 37 °C. Interestingly, the polyhydroxido species [{Pt IV (py) 2 (OH) 4 } + Na] + and [{Pt IV (py) 2 (N 3 )(OH) 3 } + Na] + resulting from the replacement of azido ligands, as determined by LC-MS, were the major products after photoirradiation of FM-190 with blue light (463 nm). This finding suggests that such photosubstituted Pt(IV) tri- and tetra-hydroxido species could play important roles in the biological activity of this anticancer complex. Density Functional Theory (DFT) and Time-Dependent DFT (TDDFT) calculations show that these Pt(IV) species arising from FM-190 in aqueous media can be formed directly from a singlet excited state. The results highlight how speciation analysis (metallomics) can shed light on photoactivation pathways for FM-190 and formation of potential excited-state pharmacophores. The ability to detect and identify photoproducts at physiologically-relevant concentrations in cells and tissues will be important for preclinical development studies of this class of photoactivatable platinum drugs.

Cow's milk is the most important dietary source of iodine in the UK and Ireland, and also contributes to dietary selenium intakes. The aim of this study was to investigate the effect of season, milk fat class (whole; semi-skimmed; skimmed) and pasteurisation on iodine and selenium concentrations in Northern Ireland (NI) milk, and to estimate the contribution of this milk to consumer iodine and selenium intakes. Milk samples (unpasteurised, whole, semi-skimmed and skimmed) were collected weekly from two large NI creameries between May 2013 and April 2014 and were analysed by inductively coupled plasma-mass spectrometry (ICP-MS). Using milk consumption data from the National Diet and Nutrition Survey (NDNS) Rolling Programme, the contribution of milk (at iodine and selenium concentrations measured in the present study) to UK dietary intakes was estimated. The mean ± standard deviation (SD) iodine concentration of milk was 475.9 ± 63.5 µg/kg and the mean selenium concentration of milk was 17.8 ± 2.7 µg/kg. Season had an important determining effect on the iodine, but not the selenium, content of cow's milk, where iodine concentrations were highest in milk produced in spring compared to autumn months (534.3 ± 53.7 vs. 433.6 ± 57.8 µg/kg, respectively; p = 0.001). The measured iodine and selenium concentrations of NI milk were higher than those listed in current UK Food Composition Databases (Food Standards Agency (FSA) (2002); FSA (2015)). The dietary modelling analysis confirmed that milk makes an important contribution to iodine and selenium intakes. This contribution may be higher than previously estimated if iodine and selenium (+25.0 and +1.1 µg/day respectively) concentrations measured in the present study were replicable across the UK at the current level of milk consumption. Iodine intakes were theoretically shown to vary by season concurrent with the seasonal variation in NI milk iodine concentrations. Routine monitoring of milk iodine concentrations is required and efforts should be made to understand reasons for fluctuations in milk iodine concentrations, in order to realise the nutritional impact to consumers., Competing Interests: The authors declare no conflict of interest.

Helicobacter pylori is a major human pathogen that is responsible for a number of gastrointestinal infections. We have used 2-DE to characterise protein synthesis in bacteria grown either on solid agar-based media or in each of two broth culture media (Brucella and brain heart infusion (BHI) broth). Significant differences were observed in the proteomes of bacteria grown either on agar-based or in broth media. Major changes in protein abundance were identified using principal component analysis (PCA), which delineated the profiles derived for the three key growth conditions (i.e. agar plates, Brucella and BHI broth). Proteins detected across the gel series were identified by peptide mass mapping and Edman sequencing. A number of proteins associated with protein synthesis in general as well as specific amino acid synthesis were depressed in broth-grown bacteria compared to plate-grown bacteria. A similar reduction was also observed in the abundance of proteins involved in detoxification. Two of the most abundant spots, identified as UreB and GroEL, in plate-grown bacteria showed a >140-fold drop in abundance in bacteria grown in Brucella broth compared to bacteria grown on agar plates. Two protein spots induced in bacteria grown in broth culture were both identified as glyceraldehyde 3-phosphate dehydrogenase based on their N-terminal amino acid sequences derived by Edman degradation. The underlying causes of the changes in the proteins abundance were not clear, but it was likely that a significant proportion of the changes were due to the alkaline pH of the broth culture media.