Characterization of Astaxanthin Nanoemulsions Produced by Intense Fluid Shear through a Self-Throttling Nanometer Range Annular Orifice Valve-Based High-Pressure Homogenizer.

Stable, oil-in-water nanoemulsions containing astaxanthin (AsX) were produced by intense fluid shear forces resulting from pumping a coarse reagent emulsion through a self-throttling annular gap valve at 300 MPa. Compared to crude emulsions prepared by conventional homogenization, a size reduction of over two orders of magnitude was observed for AsX-encapsulated oil droplets following just one pass through the annular valve. In krill oil formulations, the mean hydrodynamic diameter of lipid particles was reduced to 60 nm after only two passes through the valve and reached a minimal size of 24 nm after eight passes.

Two-Dimensional 16-BAC/SDS Polyacrylamide Gel Electrophoresis of Mitochondrial Membrane Proteins.

The substitution of the reverse polarity benzyldimethyl-n-hexadecylammonium chloride (16-BAC) polyacrylamide gel electrophoresis (PAGE) for isoelectric focusing (IEF) in the first dimension of electrophoresis improves the solubility of extremely hydrophobic proteins and their recovery compared to conventional 2D IEF/SDS PAGE. The acidic environment of 16-BAC PAGE has also been shown to better preserve the labile methylation of basic proteins such as the histones. Several improvements of the 2D 16-BAC/SDS PAGE method are collectively described here with particular emphasis on the separation of mitochondrial membrane proteins of low molecular mass.

Method for recovery and immunoaffinity enrichment of membrane proteins illustrated with metastatic ovarian cancer tissues.

Integral membrane proteins play key biological roles in cell signaling, transport, and pathogen invasion. However, quantitative clinical assays for this critical class of proteins remain elusive and are generally limited to serum-soluble extracellular fragments. Furthermore, classic proteomic approaches to membrane protein analysis typically involve proteolytic digestion of the soluble pieces, resulting in separation of intra- and extracellular segments and significant informational loss.

Comparison of in-gel protein separation techniques commonly used for fractionation in mass spectrometry-based proteomic profiling.

Fractionation of complex samples at the cellular, subcellular, protein, or peptide level is an indispensable strategy to improve the sensitivity in mass spectrometry-based proteomic profiling. This study revisits, evaluates, and compares the most common gel-based protein separation techniques i.e.

Thermal stabilization of tissues and the preservation of protein phosphorylation states for two-dimensional gel electrophoresis.

2-DE is typically capable of discriminating proteins differing by a single phosphorylation or dephosphorylation event. However, a reliable representation of protein phosphorylation states as they occur in vivo requires that both phosphatases and kinases are rapidly and completely inactivated. Thermal stabilization of mouse cerebral cortex homogenates effectively inactivated these enzymes, as evidenced by comparison with unstabilized tissues where abscissal pI shifts were a common feature in 2-D gels.

TGF-beta1 interactome: metastasis and beyond.

The ubiquitous cytokine transforming growth factor-beta1 (TGF-beta1) is one of the most potent metastatic inducers. Functional interactomic mapping using high-throughput proteomic and genomic data provides valuable insights into the regulation of tumor suppressive and metastatic attributes of TGF-beta1. Polarity changes of the TGF-beta1 interactome at a given time contributes to these contrasting effects.

Tissue fractionation by hydrostatic pressure cycling technology: the unified sample preparation technique for systems biology studies.

Major bottlenecks in systems biology studies arise from limitations of current sample preparation techniques. Multiple mutually exclusive sample preparation methods, which are often required to extract distinct classes of molecules from cells and tissues, are incompatible with studies of precious or very limited samples. Moreover, the strong detergents and chaotropic agents commonly required to solubilize sample constituents often interfere with subsequent separation and analysis.

Pilot proteomic profile of differentially regulated proteins in right atrial appendage before and after cardiac surgery using cardioplegia and cardiopulmonary bypass.

Background: Although highly protective, cardiac surgery using cardioplegia and cardiopulmonary bypass (CP/CPB) subjects myocardium to hypothermic reversible ischemic injury that can impair cardiac function which results in a greatly enhanced risk of mortality. Acute changes in myocardial contractile activity are likely regulated via protein modifications. We performed the following study to determine changes in the protein profile of human myocardium following CP/CPB.

Tris interference in IEF and 2-DE.

When dried IPGs are hydrated with protein solutions, the concentration of protein and other ionic constituents is constant throughout the strip. Tris, initially present at a very low concentration, focuses during IEF and accumulates in the gradient at a pH corresponding to its pK(a) at the operative temperature of electrophoresis. Tris focuses more rapidly than many basic proteins, and concentrates into a localized zone of increased conductivity which coincides with a precipitous voltage drop in that vicinity.

Application of pressure cycling technology to tissue sample preparation for 2-DE.

2-DE is a powerful protein analytical tool whose major strengths include semiglobal quantitation and charge separation of complex protein mixtures, enabling the analysis of differential protein expression, and variable post-translational modification. One of 2-DE's limitations relates to its limited dynamic range and consequently the number of proteins expressed that can be analyzed on a single gel. In an attempt to improve the yield of detectable proteins during sample preparation, we applied a novel extraction technique called pressure cycling technology.

Increased protein yields from Escherichia coli using pressure-cycling technology.

Sample preparation is critical to the success of two-dimensional gel electrophoresis and other analytical methods. Pressure-cycling technology (PCT) uses alternating cycles of high and low pressure to induce cell lysis. Cell suspensions were placed in PULSE Tubes and subjected to alternating cycles of high and low pressure in a Barocycler instrument.

The promise of gel-based proteomics.

First described nearly 20 years before Marc Wilkins coined the term 'proteomics', two-dimensional electrophoresis (2DGE) is still in adolescence (as is the field of proteomics). It is well recognised that two dimensions are insufficient for deconvoluting the complexity of even the simplest of proteomes, and that 2DGE can only be part of more elaborate 'multidimensional' schemes. As upstream dimensions continue to be developed, the potential of 2DGE may be further realised.

Simultaneous reduction and alkylation of protein disulfides in a centrifugal ultrafiltration device prior to two-dimensional gel electrophoresis.

Reduction and alkylation of protein disulfides prior to IEF, when performed directly in a centrifugal ultrafiltration device, provides an effective means of terminating the alkylation reaction, concentrating the proteins for analysis, and removing ionic impurities that interfere with IEF. When cells were lysed in "buffers" that support the activity of enzymes such as lysozyme and benzonase, the conductivity of the resulting lysate was an order of magnitude higher than when lysis was induced by chaotropic urea detergent solutions. Following reduction and alkylation, the conductivity of both lysates was lowered by ultrafiltration to the 0.

Solution phase isoelectric fractionation in the multi-compartment electrolyser: a divide and conquer strategy for the analysis of complex proteomes.

Sample complexity frequently interferes with the analysis of low-abundance proteins by two-dimensional gel electrophoresis (2DGE). Ideally, high abundance proteins should be removed, allowing low-abundance proteins to be applied at much higher concentrations than is possible with the unfractionated sample. One approach is to partition the sample in a manner that segregates the bulk of extraneous proteins from the protein(s) of interest.

Comparison of fluorescent stains: relative photostability and differential staining of proteins in two-dimensional gels.

The fluorescence of proteins stained with Deep Purple and SYPRO Ruby was measured over a time course of UV transillumination to determine the relative photostability of each stain. Mean spot fluorescence (n = 200 matched spots) in gels stained with Deep Purple decreased 27% following 2 min of UV transillumination, compared to SYPRO Ruby, which decreased 17%. After 19 min, an 83% decrease in Deep Purple fluorescence was observed, compared to 44% for SYPRO Ruby.