Characterisation of estrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity in the human brain

Estrogens play a crucial role in multiple functions of the brain and the proper balance of inactive estrone and active estradiol-17beta might be very important for their cerebral effects. The interconversion of estrone and estradiol-17beta in target tissues is known to be catalysed by a number of human 17beta-hydroxysteroid dehydrogenase (17beta-HSD) isoforms. The present study shows that enzyme catalysed interconversion of estrone and estradiol-17beta occurs in the human temporal lobe. The oxidative cerebral pathway preferred estradiol-17beta to Delta(5)-androstenediol and testosterone, whereas the reductive pathway preferred dehydroepiandrosterone (DHEA) to Delta(4)-androstenedione and estrone. An allosteric Hill kinetic for NAD-dependent oxidation of estradiol-17beta was observed, whereas a typical Michaelis-Menten kinetic was shown for NADPH-dependent reduction of estrone. Investigations of the interconversion of estrogens in cerebral neocortex (CX) and subcortical white matter (SC) preparations of brain tissue from 12 women and 10 men revealed no sex-differences, but provide striking evidence for the presence of at least one oxidative membrane-associated 17beta-HSD and one cytosolic enzyme that catalyses both the reductive and the oxidative pathway. Membrane-associated oxidation of estradiol-17beta was shown to be significantly higher in CX than in SC (P<0.05), whereas the cytosolic enzyme activities were significantly higher in SC than in CX (P<0.0005). Finally, real-time RT-PCR analyses revealed that besides 17beta-HSD types 4 and 5 also the isozymes type 7, 8, 10 and 11 show substantial expression in the human temporal lobe. The characteristics of the isozymes lead us to the conclusion that cytosolic 17beta-HSD type 5 is the best candidate for the observed cytosolic enzyme activities, whereas the data gave no clear answer to the question, which enzyme is responsible for the membrane-associated oxidation of estradiol-17beta. In conclusion, the study strongly suggests that different cell types and different isozymes are involved in the cerebral interconversion of estrogens, which might play a pivotal role in maintaining the functions of the central nervous system.     

A comparison of MS2-based label-free quantitative proteomic techniques with regards to accuracy and precision

The advent of algorithms for fragmentation spectrum-based label-free quantitative proteomics has enabled straightforward quantification of shotgun proteomic experiments. Despite the popularity of these approaches, few studies have been performed to assess their performance. We have therefore profiled the precision and the accuracy of three distinct relative label-free methods on both the protein and the proteome level. We derived our test data from two well-characterized publicly available quantitative data sets.     

In Vitro and in Vivo Protein-bound Tyrosine Nitration Characterized by Diagonal Chromatography

A new proteomics technique for analyzing 3-nitrotyrosine-containing peptides is presented here. This technique is based on the combined fractional diagonal chromatography peptide isolation procedures by which specific classes of peptides are isolated following a series of identical reverse-phase HPLC separation steps. Here dithionite is used to reduce 3-nitrotyrosine to 3-aminotyrosine peptides, which thereby become more hydrophilic. Our combined fractional diagonal chromatography technique was first applied to characterize tyrosine nitration in tetranitromethane-modified BSA and further led to a high quality list of 335 tyrosine nitration sites in 267 proteins in a peroxynitrite-treated lysate of human Jurkat cells. We then analyzed a serum sample of a C57BL6/J mouse in which septic shock was induced by intravenous Salmonella infection and identified six in vivo nitration events in four serum proteins, thereby illustrating that our technique is sufficiently sensitive to identify rare in vivo tyrosine nitration sites in a very complex background.Nitration of tyrosine to 3-nitrotyrosine is one of several protein modifications occurring during oxidative stress (1, 2). This modification is considered as a two-step process in which a tyrosine radical is first formed followed by the addition of ^•NO₂ yielding 3-nitrotyrosine. One of the mechanisms through which tyrosine can be nitrated is via the peroxynitrite radical (ONOO⁻); however, alternative pathways exist such as nitration by hemoperoxidases in the presence of hydrogen peroxide and nitrite (3) and reaction of the tyrosyl radical with nitric oxide yielding 3-nitrosotyrosine, which can be further oxidized to 3-nitrotyrosine.Nitration of protein-bound tyrosines can have important implications on the structure and activity of proteins (4–6) and is linked to a variety of pathological conditions such as Alzheimer disease (7) and atherosclerosis (8). Proteins containing 3-nitrotyrosine residues have mainly been identified by one- or two-dimensional PAGE followed by Western blotting using 3-nitrotyrosine-specific antibodies (9) or following affinity enrichment (10, 11). However, rather few in vivo tyrosine nitration sites have thus far been mapped onto proteins, and hence, the exact sites of in vivo nitration remain elusive. This is highly likely due to the overall low abundance of this modification as was recently illustrated by the identification of only 31 nitration sites in 29 proteins in a comprehensive analysis of mouse brain tissue covering 7,792 proteins (12). Furthermore, it was estimated that in diseased cells or organs the number of nitrated tyrosines should be as low as 0.00001–0.001% of all tyrosines (5), numbers that clearly indicate the need to enrich for 3-nitrotyrosine peptides prior to mass spectrometric analysis. In addition, several MS and MS/MS detection problems for 3-nitrotyrosine peptides were reported (13, 14) that also influence identification of such peptides.Recently, a procedure for enriching 3-nitrotyrosine peptides was described (10). In brief, reduced and alkylated proteins were digested after which primary amino groups were blocked by acetylation. Nitrotyrosines were then reduced to aminotyrosine using dithionite (15), unveiling novel primary amino groups on which sulfhydryl groups were coupled that allowed binding peptides to thiopropyl-Sepharose beads. In contrast to an earlier affinity-based isolation protocol (16), this improved enrichment procedure was more effective and led to the characterization of 150 tyrosine nitration sites in 102 proteins using a total of 3.1 mg of a mouse brain homogenate sample that was in vitro nitrated (10). However, this procedure requires many modification steps, which, when incomplete, will introduce artifacts (see “Results”). Among others, these explain the rather low number of identified nitrated tyrosines peptides using the high amount of starting material that was in vitro nitrated.Our laboratory adapted diagonal chromatography (17) for contemporary mass spectrometry-driven proteomics. Central in our combined fractional diagonal chromatography (COFRADIC1 (18)) approach is a chemical or enzymatic step that changes the reverse-phase column retention properties of a set of peptides such that these can be isolated. Among others, we developed COFRADIC protocols for isolating peptides carrying amino acid modifications such as phosphorylation (19), N-glycosylation (20), and sialylation (21) or peptides that are the result of protein processing (22–24). Here we describe a COFRADIC procedure for sorting peptides carrying nitrated tyrosines. Peptide sorting is based on a hydrophilic shift after reducing the nitro group to its amino counterpart. The applicability of COFRADIC for analyzing this modification is illustrated by characterization of four 3-nitrotyrosines in BSA treated with tetranitromethane, the mapping of 335 different nitration sites in 267 different proteins starting from 300 μg of an in vitro peroxynitrite (ONOO⁻)-treated Jurkat lysate, and the identification of six unique nitrated tyrosine residues in four serum proteins in a mouse sepsis model.     

RIBAR and xRIBAR: Methods for reproducible relative MS/MS-based label-free protein quantification

Mass spectrometry-driven proteomics is increasingly relying on quantitative analyses for biological discoveries. As a result, different methods and algorithms have been developed to perform relative or absolute quantification based on mass spectrometry data. One of the most popular quantification methods are the so-called label-free approaches, which require no special sample processing, and can even be applied retroactively to existing data sets. Of these label-free methods, the MS/MS-based approaches are most often applied, mainly because of their inherent simplicity as compared to MS-based methods. The main application of these approaches is the determination of relative protein amounts between different samples, expressed as protein ratios. However, as we demonstrate here, there are some issues with the reproducibility across replicates of these protein ratio sets obtained from the various MS/MS-based label-free methods, indicating that the existing methods are not optimally robust. We therefore present two new methods (called RIBAR and xRIBAR) that use the available MS/MS data more effectively, achieving increased robustness. Both the accuracy and the precision of our novel methods are analyzed and compared to the existing methods to illustrate the increased robustness of our new methods over existing ones.     

Redox proteomics of protein-bound methionine oxidation

We here present a new method to measure the degree of protein-bound methionine sulfoxide formation at a proteome-wide scale. In human Jurkat cells that were stressed with hydrogen peroxide, over 2000 oxidation-sensitive methionines in more than 1600 different proteins were mapped and their extent of oxidation was quantified. Meta-analysis of the sequences surrounding the oxidized methionine residues revealed a high preference for neighboring polar residues. Using synthetic methionine sulfoxide containing peptides designed according to the observed sequence preferences in the oxidized Jurkat proteome, we discovered that the substrate specificity of the cellular methionine sulfoxide reductases is a major determinant for the steady-state of methionine oxidation. This was supported by a structural modeling of the MsrA catalytic center. Finally, we applied our method onto a serum proteome from a mouse sepsis model and identified 35 in vivo methionine oxidation events in 27 different proteins.     

Combining quantitative proteomics data processing workflows for greater sensitivity

We here describe a normalization method to combine quantitative proteomics data. By merging the output of two popular quantification software packages, we obtained a 20% increase (on average) in the number of quantified human proteins without suffering from a loss of quality. Our integrative workflow is freely available through our user-friendly, open-source Rover software (http://compomics-rover.googlecode.com/).     

The dichotomy in the DNA-binding behaviour of ruthenium(II) complexes bearing benzoxazole and benzothiazole groups

The substituted tris(bipyridine)ruthenium(II) complexes {[Ru(bpy)(2)(4,4'-bbob)](2+) and [Ru(bpy)(2)(5,5'-bbob)](2+) [where bpy=2,2'-bipyridine and bbob=bis(benzoxazol-2-yl)-2,2'-bipyridine] have been prepared and compared to the previously studied complex [Ru(bpy)(2)(4,4'-bbtb)](2+) [where bbtb=bis(benzothiazol-2-yl)-2,2'-bipyridine]. From the UV/VIS titration studies, Delta-[Ru(bpy)(2)(4,4'-bbob)](2+) displays a stronger association than the Lambda-isomer with calf-thymus DNA (ct-DNA). For [Ru(bpy)(2)(5,5'-bbob)](2+), there appears to be minimal interaction with ct-DNA. The results of fluorescence titration studies suggest that [Ru(bpy)(2)(4,4'-bbob)](2+) gives an increase in emission intensity with increasing ct-DNA concentrations, with an enantiopreference for the Delta isomer, confirmed by membrane dialysis studies. The fluorescent intercalation displacement studies revealed that [Ru(bpy)(2)(4,4'-bbob)](2+) and [Ru(bpy)(2)(5,5'-bbob)](2+) display a preference for more open DNA structures such as bulge and hairpin sequences. While Lambda-[Ru(bpy)(2)(4,4'-bbtb)](2+) has shown the most significant affinity for all the oligonucleotides sequences screened in previous studies, it is the Delta isomer of the comparable benzoxazole ruthenium(II) complex (Delta-[Ru(bpy)(2)(4,4'-bbob)](2+)) that preferentially binds to DNA.     

Advances in the analysis of isothermal titration calorimetry data for ligand-DNA interactions

Isothermal titration calorimetry (ITC) is a well established technique for the study of biological interactions. The strength of ITC is that it directly measures enthalpy changes associated with interactions. Experiments can also yield binding isotherms allowing quantification of equilibrium binding constants, hence an almost complete thermodynamic profile can be established. Principles and application of ITC have been well documented over recent years, experimentally the technique is simple to use and in ideal scenarios data analysis is trivial. However, ITC experiments can be designed such that previously inaccessible parameters can be evaluated. We outline some of these advances, including (1) exploiting different experimental conditions; (2) low affinity systems; (3) high affinity systems and displacement assays. In addition we ask the question: What if data cannot be fit using the fitting functions incorporated in the data-analysis software that came with your ITC? Examples where such data might be generated include systems following non 1:n binding patterns and systems where binding is coupled to other events such as ligand dissociation. Models dealing with such data are now appearing in literature and we summarise examples relevant for the study of ligand-DNA interactions.     

Analysis of the resolution limitations of peptide identification algorithms

Proteome identification using peptide-centric proteomics techniques is a routinely used analysis technique. One of the most powerful and popular methods for the identification of peptides from MS/MS spectra is protein database matching using search engines. Significance thresholding through false discovery rate (FDR) estimation by target/decoy searches is used to ensure the retention of predominantly confident assignments of MS/MS spectra to peptides. However, shortcomings have become apparent when such decoy searches are used to estimate the FDR. To study these shortcomings, we here introduce a novel kind of decoy database that contains isobaric mutated versions of the peptides that were identified in the original search. Because of the supervised way in which the entrapment sequences are generated, we call this a directed decoy database. Since the peptides found in our directed decoy database are thus specifically designed to look quite similar to the forward identifications, the limitations of the existing search algorithms in making correct calls in such strongly confusing situations can be analyzed. Interestingly, for the vast majority of confidently identified peptide identifications, a directed decoy peptide-to-spectrum match can be found that has a better or equal match score than the forward match score, highlighting an important issue in the interpretation of peptide identifications in present-day high-throughput proteomics.     

A reproducibility-based evaluation procedure for quantifying the differences between MS/MS peak intensity normalization methods

The identification of peptides and proteins from fragmentation mass spectra is a very common approach in the field of proteomics. Contemporary high-throughput peptide identification pipelines can quickly produce large quantities of MS/MS data that contain valuable knowledge about the actual physicochemical processes involved in the peptide fragmentation process, which can be extracted through extensive data mining studies. As these studies attempt to exploit the intensity information contained in the MS/MS spectra, a critical step required for a meaningful comparison of this information between MS/MS spectra is peak intensity normalization. We here describe a procedure for quantifying the efficiency of different published normalization methods in terms of the quartile coefficient of dispersion (qcod) statistic. The quartile coefficient of dispersion is applied to measure the dispersion of the peak intensities between redundant MS/MS spectra, allowing the quantification of the differences in computed peak intensity reproducibility between the different normalization methods. We demonstrate that our results are independent of the data set used in the evaluation procedure, allowing us to provide generic guidance on the choice of normalization method to apply in a certain MS/MS pipeline application.