Protocol for the purification of proteins from biological extracts for identification by mass spectrometry

When a protein signal is selected by mass spectrometry as being a potential biomarker, it is necessary to formally identify it. This process involves separation of the protein in question and its identification by either peptide fingerprinting or tandem mass spectrometry sequencing. In the following pages, a simple and rapid protocol is described. Basically, the protocol consists of an initial rational selection of a few sorbents followed by alignment of these as a series of columns to obtain the purified target protein. This preparation is then submitted to electrophoresis, the band is excised and the trypsin digest is analyzed by either mass spectrometry (mass fingerprinting approach) or by LC-MS/MS (sequencing). The development of the process takes only a few days. Experimental data for the isolation and identification of proteins are discussed and two examples are shown.     

Sherlock Holmes and the proteome--a detective story

The performance of a hexapeptide ligand library in capturing the 'hidden proteome' is illustrated and evaluated. This library, insolubilized on an organic polymer and available under the trade name 'Equalizer Bead Technology', acts by capturing all components of a given proteome, by concentrating rare and very rare proteins, and simultaneously diluting the abundant ones. This results in a proteome of 'normalized' relative abundances, amenable to analysis by MS and any other analytical tool. Examples are given of analysis of human urine and serum, as well as cell and tissue lysates, such as Escherichia coli and Saccharomyces cerevisiae extracts. Another important application is impurity tracking and polishing of recombinant DNA products, especially biopharmaceuticals meant for human consumption.     

On the relative stabilities of the alkali cations 222 cryptates in the gas phase and in water-methanol solution

The relative stabilities of the alkali [M ⊂ 222]⁺ cryptates (M = Na, K, Rb and Cs) in the gas phase and in solution (80:20 v/v methanol:water mixture) at 298 K, are computed using a combination of ab initio quantum-chemical calculations (HF/6-31G and MP2/6-31+G*//HF/6-31+G*) and explicit-solvent Monte Carlo free-energy simulations. The results suggest that the relative stabilities of the cryptates in solution are due to a combination of steric effects (compression of large ions within the cryptand cavity), electronic effects (delocalization of the ionic charge onto the cryptand atoms) and solvent effects (dominantly the ionic dessolvation penalty). Thus, the relative stabilities in solution cannot be rationalized solely on the basis of a simple match or mismatch between the ionic radius and the cryptand cavity size as has been suggested previously. For example, although the [K ⊂ 222]⁺ cryptate is found to be the most stable in solution, in agreement with experimental data, it is the [Na ⊂ 222]⁺ cryptate that is the most stable in the gas phase. The present results provide further support to the notion that the solvent in which supramolecules are dissolved plays a key role in modulating molecular recognition processes. Figure Alkali cryptates [M ⊂ 222]+ (M = Na, K, Rb and Cs) relative stabilities in gas and methanol:water solution: solvent effects and molecular recognition

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Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Interleukin-4 inhibits caspase-3 by regulating several proteins in the Fas pathway during initial stages of human T helper 2 cell differentiation

Interleukin-4 (IL-4) is the main cytokine that polarizes activated na?ve CD4+ T cells in the T helper 2 (Th2) direction. IL-4 also regulates the subsequent stages of Th2 cell-mediated diseases, such as allergies. We conducted a proteomics study to identify IL-4-induced differences during the initial stages of T helper cell differentiation. Primary CD4+ T lymphocytes were isolated from human cord blood, activated through CD3 and CD28, and cultured in the presence or absence of IL-4. Soluble proteins were separated by two-dimensional electrophoresis and visualized by staining with autoradiography, which indicated that at least 20 proteins might be regulated by IL-4. From this minimum of 20 stained proteins, altogether 35 proteins were identified using tandem mass spectrometry. Interestingly the fragmented form of GDP dissociation inhibitor expressed in lymphocytes/Rho GDP dissociation inhibitor 2 (Ly-GDI), a known target of Caspase-3, was observed to be down-regulated in IL-4-treated cells. It was shown in further studies that IL-4 decreases Caspase-3 activity and cell death in these cells. Neutralizing Fas-Fas ligand interaction led to decreased Caspase-3 activity and lowered Ly-GDI fragmentation. We further characterized the effects of IL-4 on the expression of main regulators in the Fas-mediated pathway. We demonstrated that IL-4 decreases expression of Fas receptor and increases expression of Bid, Bcl-2, and Bcl-xL. Importantly IL-4 significantly up-regulated the short form of c-FLIP, although the levels of c-FLIP long were unaltered after IL-4 induction. Taken together, our results indicate that IL-4 inhibits caspase activity during the initial stages of human Th2 cell differentiation by regulating expression of several key players in the Fas-induced pathway.     

Independently evolving species in asexual bdelloid rotifers

Asexuals are an important test case for theories of why species exist. If asexual clades displayed the same pattern of discrete variation as sexual clades, this would challenge the traditional view that sex is necessary for diversification into species. However, critical evidence has been lacking: all putative examples have involved organisms with recent or ongoing histories of recombination and have relied on visual interpretation of patterns of genetic and phenotypic variation rather than on formal tests of alternative evolutionary scenarios. Here we show that a classic asexual clade, the bdelloid rotifers, has diversified into distinct evolutionary species. Intensive sampling of the genus Rotaria reveals the presence of well-separated genetic clusters indicative of independent evolution. Moreover, combined genetic and morphological analyses reveal divergent selection in feeding morphology, indicative of niche divergence. Some of the morphologically coherent groups experiencing divergent selection contain several genetic clusters, in common with findings of cryptic species in sexual organisms. Our results show that the main causes of speciation in sexual organisms, population isolation and divergent selection, have the same qualitative effects in an asexual clade. The study also demonstrates how combined molecular and morphological analyses can shed new light on the evolutionary nature of species.     

Comprehensive DNA signature discovery and validation

DNA signatures are nucleotide sequences that can be used to detect the presence of an organism and to distinguish that organism from all other species. Here we describe Insignia, a new, comprehensive system for the rapid identification of signatures in the genomes of bacteria and viruses. With the availability of hundreds of complete bacterial and viral genome sequences, it is now possible to use computational methods to identify signature sequences in all of these species, and to use these signatures as the basis for diagnostic assays to detect and genotype microbes in both environmental and clinical samples. The success of such assays critically depends on the methods used to identify signatures that properly differentiate between the target genomes and the sample background. We have used Insignia to compute accurate signatures for most bacterial genomes and made them available through our Web site. A sample of these signatures has been successfully tested on a set of 46 Vibrio cholerae strains, and the results indicate that the signatures are highly sensitive for detection as well as specific for discrimination between these strains and their near relatives. Our approach, whereby the entire genomic complement of organisms are compared to identify probe targets, is a promising method for diagnostic assay development, and it provides assay designers with the flexibility to choose probes from the most relevant genes or genomic regions. The Insignia system is freely accessible via a Web interface and has been released as open source software at: http://insignia.cbcb.umd.edu.     

Micropatterned biopolymer 3D scaffold for static and dynamic culture of human fibroblasts

During in vivo tissue regeneration, cell behavior is highly influenced by the surrounding environment. Thus, the choice of scaffold material and its microstructure is one of the fundamental steps for a successful in vitro culture. An efficacious method for scaffold fabrication should prove its versatility and the possibility of controlling micro- and nanostructure. In this paper, hyaluronic acid 3D scaffolds were developed through lamination of micropatterned membranes, fabricated after optimization of a soft-lithography method. The scaffold presented here is characterized by a homogeneous hexagonal lattice with porosity of 69%, specific surface area of 287 cm-1, and permeability of 18.9 microm2. The control over the geometry was achieved with an accuracy of 20 mum. This technique allowed not only fabrication of planar 3D scaffolds but also production of thin wall tubular constructs. Mechanical tests, performed on dry tubular scaffolds, show high rupture tensile strength. This construct could be promising not only as engineered vascular grafts but also for regeneration of skin, urethra, and intestinal walls. The biocompatibility of a 3D planar scaffold was tested by seeding human fibroblasts. The cells were cultured in both static and dynamic conditions, in a perfusion bioreactor at different flow rates. Microscope analysis and MTT test showed cell proliferation and viability and a uniform cell distribution likely due to an appropriate lattice structure.     

Interspecific hybridization in natural sturgeon populations of the Eastern Black Sea: the consequence of drastic population decline?

The eastern part of the Black Sea and its tributaries are suitable habitats for several sturgeon species, among which Acipenser gueldenstaedtii, A. stellatus, A. nudiventris, A. persicus, A. sturio, and H. huso are well documented. However, different threats have led these species to a dramatic decline, all of them are currently listed as Critically Endangered, and some Locally Extinct, in that area. We tested 94 wild sturgeon samples from the Black Sea and Rioni River by analyzing the mitochondrial Control Region and nuclear markers for hybrid identification. The data analyses (1) assessed mitochondrial diversity among samples, (2) identified their species, as well as (3) indicated instances of hybridization. The data collected, besides confirming a sharp decrease of catches of Beluga and Stellate sturgeon in recent years, also revealed four juvenile hybrids between Russian and Stellate sturgeon, providing the first evidence of natural interspecific hybridization in the Rioni. The present communication raises concerns about the status of sturgeon species in this area and underlines the urgent need for conservation programs to restore self-sustaining populations.

     

Protein kinase D1 inhibition interferes with mitosis progression

Protein kinase D1 (PKD1) plays a vital role in signal transduction, cell proliferation, membrane trafficking, and cancer; however, the majority of the studies up to date had centered primarily on PKD1 functions in interphase, very little is known about its role during cell division. We previously demonstrated that during mitosis PKD1 is activated and associated with centrosomes, spindles, and midbodies. However, these observations did not address whether PKD1 was associated with mitosis regulation. Accordingly, we used rapidly acting PKD-specific inhibitors to examine the contribution of PKD1 the sequence of events in mitosis. We found that although PKD1 overexpression did not affect mitosis progression, suppression of its catalytic activity by two structurally unrelated inhibitors (kb NB 142-70 and CRT 0066101) induced a significant delay in metaphase to anaphase transition time. PKD1 inhibition during mitosis also produced the appearance of abnormal spindles, defects in chromosome alignment, and segregation as well as apoptosis. Thus, these observations indicate that PKD1 activity is associated with mitosis regulation.     

p53 Represses the Mevalonate Pathway to Mediate Tumor Suppression

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