Dried Blood Spot Proteomics: Surface Extraction of Endogenous Proteins Coupled with Automated Sample Preparation and Mass Spectrometry Analysis

Dried blood spots offer many advantages as a sample format including ease and safety of transport and handling. To date, the majority of mass spectrometry analyses of dried blood spots have focused on small molecules or hemoglobin. However, dried blood spots are a potentially rich source of protein biomarkers, an area that has been overlooked. To address this issue, we have applied an untargeted bottom-up proteomics approach to the analysis of dried blood spots. We present an automated and integrated method for extraction of endogenous proteins from the surface of dried blood spots and sample preparation via trypsin digestion by use of the Advion Biosciences Triversa Nanomate robotic platform. Liquid chromatography tandem mass spectrometry of the resulting digests enabled identification of 120 proteins from a single dried blood spot. The proteins identified cross a concentration range of four orders of magnitude. The method is evaluated and the results discussed in terms of the proteins identified and their potential use as biomarkers in screening programs.

A concise asymmetric synthesis of cis-2,6-disubstituted N-aryl piperazines via Pd-catalyzed carboamination reactions

A concise, modular, asymmetric synthesis of cis-2,6-disubstituted piperazines from readily available amino acid precursors is described. The key step in the synthesis is a Pd-catalyzed carboamination of a N1-aryl-N2-allyl-1,2-diamine with an aryl bromide. The products are obtained in 14-20:1 dr, with >97% ee, and the key cyclizations are the first examples of six-membered ring formation via Pd-catalyzed carboamination reactions of unsaturated amines with aryl halides.     

A general modular method of azaindole and thienopyrrole synthesis via Pd-catalyzed tandem couplings of gem-dichloroolefins

A palladium-catalyzed reaction of gem-dichloroolefins and a boronic acid via a tandem intramolecular C-N and intermolecular Suzuki coupling process gave corresponding substituted azaindoles or thienopyrroles. This method is a very modular protocol to synthesize all four isomers of azaindole and two isomers of thienopyrroles in good to excellent yield.     

Specific and genotypic identification of Cryptosporidium from a broad range of host species by nonisotopic SSCP analysis of nuclear ribosomal DNA

The accurate identification of Cryptosporidium (Protozoa: Apicomplexa) species and genotypes is central to the understanding of the transmission and to the diagnosis and control of cryptosporidiosis. In this study, we demonstrate the effectiveness of nonisotopic SSCP analysis of a approximately 300 bp region of the small subunit (pSSU) of ribosomal DNA for the specific identification of and delineation among 18 different Cryptosporidium species and genotypes from a wide range of hosts. This mutation scanning approach allowed the rapid and reliable differentiation between species/genotypes differing by as little as 1.3% in the pSSU sequence, with the capacity to detect point mutations. The present findings confirm the usefulness of this tool for the rapid genetic screening of Cryptosporidium samples from any host species, providing a foundation for detailed systematic, epidemiological and ecological studies. Although applied herein to pSSU, this low cost approach should be applicable to a wide range of genetic loci for population genetic investigations of Cryptosporidium.     

Top-Down and Bottom-Up Identification of Proteins by Liquid Extraction Surface Analysis Mass Spectrometry of Healthy and Diseased Human Liver Tissue

Liquid extraction surface analysis mass spectrometry (LESA MS) has the potential to become a useful tool in the spatially-resolved profiling of proteins in substrates. Here, the approach has been applied to the analysis of thin tissue sections from human liver. The aim was to determine whether LESA MS was a suitable approach for the detection of protein biomarkers of nonalcoholic liver disease (nonalcoholic steatohepatitis, NASH), with a view to the eventual development of LESA MS for imaging NASH pathology. Two approaches were considered. In the first, endogenous proteins were extracted from liver tissue sections by LESA, subjected to automated trypsin digestion, and the resulting peptide mixture was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) (bottom-up approach). In the second (top-down approach), endogenous proteins were extracted by LESA, and analyzed intact. Selected protein ions were subjected to collision-induced dissociation (CID) and/or electron transfer dissociation (ETD) mass spectrometry. The bottom-up approach resulted in the identification of over 500 proteins; however identification of key protein biomarkers, liver fatty acid binding protein (FABP1), and its variant (Thr→Ala, position 94), was unreliable and irreproducible. Top-down LESA MS analysis of healthy and diseased liver tissue revealed peaks corresponding to multiple (~15–25) proteins. MS/MS of four of these proteins identified them as FABP1, its variant, α-hemoglobin, and 10 kDa heat shock protein. The reliable identification of FABP1 and its variant by top-down LESA MS suggests that the approach may be suitable for imaging NASH pathology in sections from liver biopsies. Graphical Abstract

?     

Anti-diabetic effects of a series of vanadium dipicolinate complexes in rats with streptozotocin-induced diabetes

The effects of oral treatment of rats with streptozotocin-induced diabetes with a range of vanadium dipicolinate complexes (Vdipic) and derivatives are reviewed. Structure–reactivity relationships are explored aiming to correlate properties such as stability, to their insulin-enhancing effects. Three types of modifications are investigated; first, substitutions on the aromatic ring, second, coordination of a hydroxylamido group to the vanadium, and third, changes in the oxidation state of the vanadium ion. These studies allowed us to address the importance of coordination chemistry, and redox chemistry, as modes of action. Dipicolinate was originally chosen as a ligand because the dipicolinatooxovanadium(V) complex (V5dipic), is a potent inhibitor of phosphatases. The effect of vanadium oxidation state (3, 4 or 5), on the insulin-enhancing properties was studied in both the Vdipic and VdipicCl series. Effects on blood glucose, body weight, serum lipids, alkaline phosphatase and aspartate transaminase were selectively monitored. Statistically distinct differences in activity were found, however, the trends observed were not the same in the Vdipic and VdipicCl series. Interperitoneal administration of the Vdipic series was used to compare the effect of administration mode. Correlations were observed for blood vanadium and plasma glucose levels after V5dipic treatment, but not after treatment with corresponding V4dipic and V3dipic complexes. Modifications of the aromatic ring structure with chloride, amine or hydroxyl groups had limited effects. Global gene expression was measured using Affymetrix oligonucleotide chips. All diabetic animals treated with hydroxyl substituted V5dipic (V5dipicOH) and some diabetic rats treated with vanadyl sulfate had normalized hyperlipidemia yet uncontrolled hyperglycemia and showed abnormal gene expression patterns. In contrast to the normal gene expression profiles previously reported for some diabetic rats treated with vanadyl sulfate, where both hyperlipidemia and hyperglycemia were normalized. Modification of the metal, changing the coordination chemistry to form a hydroxylamine ternary complex, had the most influence on the anti-diabetic action. Vanadium absorption into serum was determined by atomic absorption spectroscopy for selected vanadium complexes. Only diabetic rats treated with the ternary V5dipicOH hydroxylamine complex showed statistically significant increases in accumulation of vanadium into serum compared to diabetic rats treated with vanadyl sulfate. The chemistry and physical properties of the Vdipic complexes correlated with their anti-diabetic properties. Here, we propose that compound stability and ability to interact with cellular redox reactions are key components for the insulin-enhancing activity of vanadium compounds. Specifically, we found that the most overall effective anti-diabetic Vdipic compounds were obtained when the compound administered had an increased coordination number in the vanadium complex.     

Palladium-catalyzed alkene carboamination reactions for the synthesis of substituted piperazines

A strategy for the stereoselective preparation of enantiomerically enriched cis-2,6-disubstituted piperazines from amino acid precursors is described. The target compounds are generated in 95-99% ee with good to excellent levels of diastereoselectivity (usually 14:1 to >20:1) using Pd-catalyzed carboamination reactions between aryl or alkenyl halides and substituted ethylenediamine derivatives to form the heterocyclic rings. The synthesis requires only 4-5 steps from commercially available amino acids, and allows for the modular construction of piperazines bearing different substituents at N¹, N⁴, C², and C⁶. The use of this strategy for the construction of 2,3-disubstituted piperazines, fused bicyclic piperazines, and tetrahydroquinoxalines is also reported. In addition, the mechanism of the key carboamination reactions is discussed, and new models that predict and explain the stereochemical outcome of these transformations are presented.