Fully denaturing two-dimensional electrophoresis of membrane proteins: a critical update

The quality and ease of proteomics analysis depends on the performance of the analytical tools used, and thus of the performances of the protein separation tools used to deconvolute complex protein samples. Among protein samples, membrane proteins are one of the most difficult sample classes, because of their hydrophobicity and embedment in the lipid bilayers. This review deals with the recent progresses and advances made in the separation of membrane proteins by 2-DE separating only denatured proteins. Traditional 2-D methods, i.e., methods using IEF in the first dimension are compared to methods using only zone electrophoresis in both dimensions, i.e., electrophoresis in the presence of cationic or anionic detergents. The overall performances and fields of application of both types of method is critically examined, as are future prospects for this field

Development of Container Free Sample Exposure for Synchrotron X-ray Footprinting.

The method of X-ray footprinting and mass spectrometry (XFMS) on large protein assemblies and membrane protein samples requires high flux density to overcome the hydroxyl radical scavenging reactions produced by the buffer constituents and the total protein content. Previously, we successfully developed microsecond XFMS using microfluidic capillary flow and a microfocused broadband X-ray source at the Advanced Light Source synchrotron beamlines, but the excessive radiation damage incurred when using capillaries prevented the full usage of a high-flux density beam. Here we present another significant advance for the XFMS method: the instrumentation of a liquid injection jet to deliver container free samples to the X-ray beam. Our preliminary experiments with a liquid jet at a bending magnet X-ray beamline demonstrate the feasibility of the approach and show a significant improvement in the effective dose for both the Alexa fluorescence assay and protein samples compared to conventional capillary flow methods. The combination of precisely controlled high dose delivery, shorter exposure times, and elimination of radiation damage due to capillary effects significantly increases the signal quality of the hydroxyl radical modification products and the dose-response data. This new approach is the first application of container free sample handling for XFMS and opens up the method for even further advances, such as high-quality microsecond time-resolved XFMS studies

FTIR-based prediction of collagen content in hydrolyzed protein samples

publishedVersio

Identification of the alternative oxidase gene and its expression in the copepod Tigriopus californicus

In addition to the typical electron transport system (ETS) in animal mitochondria responsible for oxidative phosphorylation, in some species there exists an alternative oxidase (AOX) pathway capable of catalyzing the oxidation of ubiquinol and the reduction of oxygen to water. The discovery of AOX in animals is recent and further investigations into its expression, regulation, and physiological role have been hampered by the lack of a tractable experimental model organism. Our recent DNA database searches using bioinformatics revealed an AOX sequence in several marine copepods including Tigriopus californicus. This species lives in tidepools along the west coast of North America and is subject to a wide variety of daily environmental stresses. Here we verify the presence of the AOX gene in T. californicus and the expression of AOX mRNA and AOX protein in various life stages of the animal. We demonstrate that levels of the AOX protein increase in T. californicus in response to cold and heat stress compared to normal rearing temperature. We predict that a functional AOX pathway is present in T. californicus, propose that this species will be a useful model organism for the study of AOX in animals, and discuss future directions for animal AOX research

Top-down analysis of protein samples by de novo sequencing techniques

Motivation: Recent technological advances have made high-resolution mass spectrometers affordable to many laboratories, thus boosting rapid development of top-down mass spectrometry, and implying a need in efficient methods for analyzing this kind of data. Results: We describe a method for analysis of protein samples from top-down tandem mass spectrometry data, which capitalizes on de novo sequencing of fragments of the proteins present in the sample. Our algorithm takes as input a set of de novo amino acid strings derived from the given mass spectra using the recently proposed Twister approach, and combines them into aggregated strings endowed with offsets. The former typically constitute accurate sequence fragments of sufficiently well-represented proteins from the sample being analyzed, while the latter indicate their location in the protein sequence, and also bear information on post-translational modifications and fragmentation patterns. Availability and Implementation: Freely available on the web at http://bioinf.spbau.ru/en/twister

Lipid storage and autophagy in melanoma cancer cells

Cancer stem cells (CSC) represent a key cellular subpopulation controlling biological features such as cancer progression in all cancer types. By using melanospheres established from human melanoma patients, we compared less differentiated melanosphere-derived CSC to differentiating melanosphere-derived cells. Increased lipid uptake was found in melanosphere-derived CSC vs. differentiating melanosphere-derived cells, paralleled by strong expression of lipogenic factors Sterol Regulatory Element-Binding Protein-1 (SREBP-1) and Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ). An inverse relation between lipid-storing phenotype and autophagy was also found, since microtubule-associated protein 1A/1B-Light Chain 3 (LC3) lipidation is reduced in melanosphere-derived CSC. To investigate upstream autophagy regulators, Phospho-AMP activated Protein Kinase (P-AMPK) and Phospho-mammalian Target of Rapamycin (P-mTOR) were analyzed; lower P-AMPK and higher P-mTOR expression in melanosphere-derived CSC were found, thus explaining, at least in part, their lower autophagic activity. In addition, co-localization of LC3-stained autophagosome spots and perilipin-stained lipid droplets was demonstrated mainly in differentiating melanosphere-derived cells, further supporting the role of autophagy in lipid droplets clearance. The present manuscript demonstrates an inverse relationship between lipid-storing phenotype and melanoma stem cells differentiation, providing novel indications involving autophagy in melanoma stem cells biology

Effect of isolation conditions on structural properties and surface behavior of soy-whey proteins

In this study, the impact of isolation conditions on structural and surface properties at the air/water interface of soy-whey proteins (SWP) was assessed. SWP were obtained by precipitation of soy-whey (at pH 4.5 or 8.0) with acetone or ammonium sulfate. Despite the fact that all SWP samples exhibited similar electrophoretic patterns, they showed different protein content (from 54.2 to 98.2% w/w). When precipitation was performed at pH 4.5, SWP samples evidenced a decrease of protein solubility (SP) and thermal stability, while the precipitation with acetone promoted the enrichment in polysaccharides and minerals. For all samples, intrinsic fluorescence, surface hydrophobicity and Fourier transform infrared (FTIR) studies revealed structural changes correlated to protein unfolding and aggregation processes. However, the surface behavior can be predicted from these studies mainly due to differences in surface hydrophobicity and the differential contribution of insoluble aggregates. The heating of SWP samples enhanced the surface activity, regardless of the pH of the raw material and the isolation method. These results can be useful as a reference research and as a starting point for industrial exploitation of proteins from soy wastewater.Fil: Ingrassia, Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Física de Rosario. Universidad Nacional de Rosario. Instituto de Física de Rosario; ArgentinaFil: Sobral, Pablo Antonio. Universidad Nacional de La Plata. Facultad de Ciencias Exactas; ArgentinaFil: Risso, Patricia Hilda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Física de Rosario. Universidad Nacional de Rosario. Instituto de Física de Rosario; ArgentinaFil: Palazolo, Gonzalo Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Funcionalidad y Tecnología de Alimentos; ArgentinaFil: Wagner, Jorge Ricardo. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Funcionalidad y Tecnología de Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin