Quality control in LC-MS/MS

In the last 15 years, MS-based protein characterization has expanded at a rapid rate. This success is built upon constantly improving instrumentation and a variety of ingenious methods applied to numerous biological questions. However, the reproducibility of mass spectrometric results is considered by many as insufficient. In part, inadequate quality control might be responsible for the lack of reproducibility. Quality control is rarely discussed in scientific publications. Here, we briefly present measures undertaken in our laboratory to foster a general discussion of the subject.     

The influence of Aspalathus linearis (Rooibos) and dihydrochalcones on adrenal steroidogenesis: quantification of steroid intermediates and end products in H295R cells

The steroid hormone output of the adrenal gland is crucial in the maintenance of hormonal homeostasis, with hormonal imbalances being associated with numerous clinical conditions which include, amongst others, hypertension, metabolic syndrome, cardiovascular disease, insulin resistance and type 2 diabetes. Aspalathus linearis (Rooibos), which has been reported to aid stress-related symptoms linked to metabolic diseases, contains a wide spectrum of bioactive phenolic compounds of which aspalathin is unique. In this study the inhibitory effects of Rooibos and the dihydrochalcones, aspalathin and nothofagin, were investigated on adrenal steroidogenesis. The activities of both cytochrome P450 17α-hydroxylase/17,20 lyase and cytochrome P450 21-hydroxylase were significantly inhibited in COS-1 cells. In order to study the effect of these compounds in H295R cells, a human adrenal carcinoma cell line, a novel UPLC-MS/MS method was developed for the detection and quantification of twenty-one steroid metabolites using a single chromatographic separation. Under both basal and forskolin-stimulated conditions, the total amount of steroids produced in H295R cells significantly decreased in the presence of Rooibos, aspalathin and nothofagin. Under stimulated conditions, Rooibos decreased the total steroid output 4-fold and resulted in a significant reduction of aldosterone and cortisol precursors. Dehydroepiandrosterone-sulfate levels were unchanged, while the levels of androstenedione (A4) and 11β-hydroxyandrostenedione (11βOH-A4) were inhibited 5.5 and 2.3-fold, respectively. Quantification of 11βOH-A4 showed this metabolite to be a major product of steroidogenesis in H295R cells and we confirm, for the first time, that this steroid metabolite is the product of the hydroxylation of A4 by human cytochrome P450 11β-hydroxylase. Taken together our results demonstrate that Rooibos, aspalathin and nothofagin influence steroid hormone biosynthesis and the flux through the mineralocorticoid, glucocorticoid and androgen pathways, thus possibly contributing to the alleviation of negative effects arising from elevated glucocorticoid levels.     

Density Functional Study on UV/VIS Spectra of Copper-Protein Active Sites: The Effect of Mutations

UV/VIS Electron excitation spectra have been computed for large, realistic model systems of the blue copper protein family. Fully quantum-chemical calculations at the density-functional theory level employing polarized triple-ζ basis sets have been performed on systems of over 120 atoms, without symmetry. Different mutants, with the ligating methionine of the wild type Cu center exchanged for histidine (M121?H) and glutamine (M121Q), have been investigated in order to obtain insight about how the influence of the exact surrounding milieu of the Cu-atom affects the computed spectrum. With sufficiently large model sizes, inclusion of the environment by using continuum solvation models do not change the spectra significantly. More direct and rigorous treatments are needed to reliably assess the effect of the surrounding protein on the electronic structure of the active sites.     

Effects of dehydration on cardiovascular development in the embryonic American alligator (Alligator mississipiensis)

Effects of dehydration on reptilian embryonic cardiovascular function are unknown. Here, we present the first morphological and physiological data quantifying the cumulative effects of four acute dehydration events on the embryonic American alligator, Alligator mississipiensis. We hypothesized that dehydration would alter embryonic morphology, reduce blood volume and augment the response to angiotensin II (Ang II), a key osmotic and blood volume regulatory response element in adult vertebrates. Drying events at 30%, 40%, 50%, and 60% of embryonic incubation reduced total egg water content by 14.43 ± 0.37 g, a 3.4 fold increase relative to controls. However, embyronic blood volume was greater in the dehydration group at 70% of embryonic incubation compared to controls (0.39 ± 0.044 mLg(-1) and 0.22 ± 0.03 mLg(-1), respectively), however, both groups were similar at 90% of incubation (0.18 ± 0.02 mLg(-1) in the controls and 0.23 ± 0.03 mLg(-1) in the dehydrated group). Dehydration altered the morphological phenotype and resulted in an overall reduction in embryonic mass at both incubation time points measured. Dehydration also altered the physiological phenotype, resulting in embryonic alligators that were relatively bradycardic at 90% of incubation. Arterial Ang II injections resulted in a dose dependent hypertension, which increased in intensity over the span of incubation studied. While progressive incubation altered the Ang II response, dehydration had no impact on the cardiovascular responses to the peptide. Quantification of Ang II type-1 receptor protein using western blot analysis illustrated that dehydration condition and incubation time point did not alter protein quantity. Collectively, our results show that dehydration during embryonic development of the American alligator alters embryonic morphology and baseline heart rate without altering arterial pressure and response to Ang II.     

Effect of Ga3+ and Gd3+ ions substitution on the structural and optical properties of Ce3+‐doped yttrium aluminium garnet phosphor nanopowders

The structural and optical properties of commercially obtained Y₃Al₅O₁₂:Ce³⁺ phosphor were investigated by replacing Al³⁺ with Ga³⁺ and Y³⁺ with Gd³⁺ in the Y₃Al₅O₁₂:Ce³⁺ structure to form Y₃(Al,Ga)₅O₁₂:Ce³⁺ and (Y,Gd)₃Al₅O₁₂:Ce³⁺. X‐Ray diffraction (XRD) results showed slight 2‐theta peak shifts to lower angles when Ga³⁺ was used and to higher angles when Gd³⁺ was used, with respect to peaks from Y₃Al₅O₁₂:Ce³⁺ and JCPDS card no. 73–1370. This could be attributed to induced crystal‐field effects due to the different ionic sizes of Ga³⁺ and Gd³⁺ compared with Al³⁺ and Y³⁺. The photoluminescence (PL) spectra showed broad excitation from 350 to 550 nm with a maximum at 472 nm, and broad emission bands from 500 to 650 nm, centred at 578 nm for Y₃Al₅O₁₂:Ce³⁺ arising from the 5d → 4f transition of Ce³⁺. PL revealed a blue shift for Ga³⁺ substitution and a red shift for Gd³⁺ substitution. UV–Vis showed two absorption peaks at 357 and 457 nm for Y₃Al₅O₁₂:Ce³⁺, with peaks shifting to 432 nm for Ga³⁺ and 460 nm for Gd³⁺ substitutions. Changes in the trap levels or in the depth and number of traps due to Ce³⁺ were analysed using thermoluminescence (TL) spectroscopy. This revealed the existence of shallow and deep traps. It was observed that Ga³⁺ substitution contributes to the shallowest traps at 74 °C and fewer deep traps at 163 °C, followed by Gd³⁺ with shallow traps at 87 °C and deep traps at 146 °C. Copyright © 2016 John Wiley & Sons, Ltd.     

Evidence for the functional role of residues in the B'-C loop of baboon cytochrome P450 side-chain cleavage (CYP11A1) obtained by site-directed mutagenesis, kinetic analysis and homology modelling

To gain further insight into the structure/function relationship of cytochrome P450 side-chain cleavage (CYP11A1), this enzyme was investigated in the Cape baboon (Papio ursinus). Four constructs were cloned and characterised in non-steroidogenic mammalian COS-1 cells. Wild type recombinant baboon CYP11A1 cDNA yielded a K(m) value of 1.6 microM for 25-hydroxycholesterol. The single amino acid substitutions, I98Q and I98K resulted in a 1.7- and 2.8-fold increases in K(m) values, respectively. Conversely, the introduction of the mutation, K103A, resulted in a 1.8-fold decrease in K(m). A homology model of CYP11A1, based on the crystal structures of CYP102 and CYP2C5, revealed that residues 98 and 103 lie within the B'-C loop and contribute to the spatial orientation and structural integrity of this domain. Based on these results we propose a topological model of the CYP11A1 active pocket, which is supported by substrate docking analysis and kinetic studies.     

Growth and Mating of <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> var. <Emphasis Type="Italic">grubii</Emphasis> on Woody Debris

A total of 36 Cryptococcus neoformans strains originating from South Africa were screened for wood degrading enzymes. All strains tested positive for cellulase activity while none where capable of xylan degradation. Three C. neoformans var. grubii strains, originating from clinical and environmental samples, representing the same genotype (VNI/AFLP1—C. neoformans var. grubii) and MATα, were evaluated for growth on debris of two common tree species in South Africa: Acacia mearnsii and Eucalyptus camaldulensis. The mating capability of all the C. neoformans strains was evaluated on similar debris. Strains grown on A. mearnsii yielded substantially greater yeast populations. A total of 26%, 6%, 46%, and 80% of the 36 C. neoformans strains tested were either able to mate or develop filaments when crossed on A. mearnsii and E. camaldulensis debris, V8 juice, and yeast carbon base (YCB) agar, respectively. Filamentation and monokaryotic fruiting was observed in 3% of strains when C. neoformans was cultured on either A. mearnsii, E. camaldulensis debris, or YCB. The results indicate that this fungus is capable of completing its life cycle and can produce basidiospores on woody debris. In the future, these findings should be considered when studying the epidemiology, microbial ecology, and proposed infection process of this global pathogen.     

The effects of palm oil breakdown products on lipid turnover and morphology of fungi

The oleaginous fungi Cryptococcus curvatus and Mucor circinelloides were used to determine the effect of palm oil breakdown products, measured as polymerized triglycerides (PTGs), on lipid turnover and on fungal growth and morphology. In M. circinelloides, we found after 7 days of growth, a decrease in biomass and in lipid utilization and accumulation at increased PTG levels, both at low and neutral pH. In C.?curvatus, there was also a decrease in lipid utilization and biomass production at increased PTG levels, at both low and neutral pH. However, an increase in oil accumulation was observed at low pH while it remained similar at neutral pH for all PTG levels tested. Hairy and warty protuberances on the cell surface were observed when C. curvatus was grown on oils with 15% and 45% PTGs, respectively. Using nano scanning Auger microscopy, we found no evidence to suggest a difference in elemental composition of the surfaces of the warty protuberances compared with the rest of the cell wall surface. We conclude that the warty protuberances are outgrowths of cell walls and that the changes observed in lipid turnover in both fungi are due to the presence of palm oil breakdown products.     

Differential stabilization of adenine quartets by anions and cations

We have investigated the structures and stabilities of four different adenine quartets with alkali and halide ions in the gas phase and in water, using dispersion-corrected density functional theory at the BLYP-D/TZ2P level. First, we examine the empty quartets and how they interact with alkali cations and halide anions with formation of adenine quartet–ion complexes. Second, we examine the interaction in a stack, in which a planar adenine quartet interacts with a cation or anion in the periphery as well as in the center of the quartet. Interestingly, for the latter situation, we find that both cations and anions can stabilize a planar adenine quartet in a stack.