Improved Method for Virus Structural Polypeptide Analysis on Dissociating Acylamide Gel

A method is described in which polypeptides can be separated, with a high band resolution, by electrophoresis through "pore gradient" acrylamide gel (15 cm in length) in a sodium dodecyl sulfate-urea dissociating system. The applicability of this technique to the analysis of virus structural components was examined with the adenovirus type 2 model system.     

Resolution and Reconstitution of a Mammalian Membrane

The problem of the resolution and reconstitution of the inner mitochondrial membrane has been approached at three levels. (1) Starting with phosphorylating submitochondrial particles, a "resolution from without" can be achieved by stripping of surface components. The most extensive resolution was recently obtained with the aid of silicotungstate. Such particles require for oxidative phosphorylation the addition of several coupling factors as well as succinate dehydrogenase. (2) Starting with submitochondrial particles that have been degraded by trypsin and urea a resolution of the inner membrane proper containing an ATPase has been achieved. These experiments show that at least five components are required for the reconstitution of an oligomycin-sensitive ATPase: a particulate component, F ₁, Mg⁺⁺, phospholipids, and F_c. Morphologically, the reconstituted ATPase preparations resemble submitochondrial particles. (3) Starting with intact mitochondria individual components of the oxidation chain have been separated from each other. The following components were required for the reconstitution of succinoxidase: succinate dehydrogenase, cytochrome b\, cytochrome c ₁, cytochrome c, cytochrome oxidase, phospholipids and Q ₁₀. The reconstituted complex had properties similar to those of phosphorylating submitochondrial particles; i.e., the oxidation of succinate by molecular oxygen was highly sensitive to antimycin.     

Chromatography of rare basic amino acids

l-Ornithine, l-lysine, α,γ-diaminobutyric acid, and α,β-diaminopropionic acid were separated chromatographically on PA 35 in the presence of amino acids normally present in protein hydrolysates or physiological fluids. The resolution of these components in the mixture was excellent. The content of these lower homologs of lysine can be quantitatively determined by means of the chromatography.     

The fractionation of high-molecular-weight ribonucleic acid by polyacrylamide-gel electrophoresis

1. Gels were prepared with recrystallized acrylamide and bisacrylamide. Electrophoresis was in tris-sodium acetate-EDTA buffer for 0.5 to 3hr. Gels were scanned at 280 or 265mmu. Techniques are described for slicing and radioactive counting. 2. The mobility of RNA was inversely related to the sedimentation coefficient and varied with gel concentration. Electrophoresis in 2.2-2.6% gels gives a fractionation similar to density-gradient centrifugation. It shows the two ribosomal RNA components, the 45s precursor, transfer RNA and minor components. In 5% and 7.5% gels, 4s and 5s RNA separated and ribosomal RNA was excluded. 3. The resolution is greater and more detailed than by centrifugation, and many samples can be analysed simultaneously and rapidly.     

Separation and quantitation of molecular species from plant lipids by high-performance liquid chromatography

Monogalactosyl-, digalactosyl-, and sulfoquinovosyl diacylglycerol as well as phosphatidyl glycerol were isolated by conventional TLC and then separately subjected to HPLC for resolution of molecular species. Molecular species emerge in groups from reversed-phase columns during gradient elution. The groups are separated according to the sum of carbon and double bond numbers in fatty acyl pairs in linear relation to elution times. Therefore, it is possible to identify a species group with respect to carbon and double bond numbers by its retention time. The separation is monitored by recording the absorbance at 200 nm which depends on double bond combinations in acyl pairs. Diacylglycerols released from glyco- and phospholipids were separated as rho-anisoyl derivatives according to similar criteria. In this case separation was monitored at 250 nm, at which wavelength the absorbance is directly related to molar proportions. By calculating corrected 200-nm/250-nm absorbance ratios for different molecular species of rho-anisoyl diacylglycerols, relative response factors for different double bond combinations were obtained. The 200-nm absorbances of intact lipid species can be converted to molar proportions by division with these factors.     

The hyperactivity of hamster fibroblast lysosomal enzymes after endocytosis of sucrose.

The sialoglycoproteins of the human erythrocyte membrane can be separated into at least eight periodic acid Schiff-positive components using sodium dodecyl sulfate gel electrophoresis in the discontinuous buffer system of Laemmli. All eight components can also be labeled by mild periodate oxidation followed by reduction with NaB³H₄. Periodic acid Schiff 2 can be resolved into at least three distinct components. Two sialoglycoproteins, with apparent molecular weights of 35,000 and 27,000 do not appear to be labeled by lactoperoxidase-catalyzed iodination at the external membrane surface.     

The structure of the gamma-tubulin small complex: implications of its architecture and flexibility for microtubule nucleation

The gamma-tubulin small complex (gamma-TuSC) is an evolutionarily conserved heterotetramer essential for microtubule nucleation. We have determined the structure of the Saccharomyces cerevisiae gamma-TuSC at 25-A resolution by electron microscopy. gamma-TuSC is Y-shaped, with an elongated body connected to two arms. Gold labeling showed that the two gamma-tubulins are located in lobes at the ends of the arms, and the relative orientations of the other gamma-TuSC components were determined by in vivo FRET. The structures of different subpopulations of gamma-TuSC indicate flexibility in the connection between a mobile arm and the rest of the complex, resulting in variation of the relative positions and orientations of the gamma-tubulins. In all of the structures, the gamma-tubulins are distinctly separated, a configuration incompatible with the microtubule lattice. The separation of the gamma-tubulins in isolated gamma-TuSC likely plays a role in suppressing its intrinsic microtubule-nucleating activity, which is relatively weak until the gamma-TuSC is incorporated into higher order complexes or localized to microtubule-organizing centers. We propose that further movement of the mobile arm is required to bring the gamma-tubulins together in microtubule-like interactions, and provide a template for microtubule growth.     

Analysis of plasmid samples on a microchip

We have developed a LabChip-based plasmid assay that runs on the Agilent 2100 Bioanalyzer. The assay determines the sizes and relative concentrations of the multiple forms of plasmid samples. Twelve samples can be analyzed on each chip in an automated run lasting approximately 30min. By using a supercoiled DNA sizing standard of 2-16kb, the size of the analyzed plasmid can be determined. The resulting MW has a relative standard deviation (CV) <5% and error <5%. Plasmids from 2-8kb can be separated with resolution better than 1kb. Topological isoforms in a plasmid sample can also be separated. However, due to differential staining, the heterogeneity of plasmid samples can only be measured if the signal of each isomer peak can be calibrated with pure standards for every isomer form. For a typical plasmid preparation which predominately is in the supercoiled form, the normalized corrected peak area for the supercoiled form correlates with the plasmid concentration in a broad range of 1-100ng/microl. The measurement is semiquantitative with a CV lower than 20%. A number of applications of this assay on a Labchip will be shown.     

Pulsed homogeneous orthogonal field gel electrophoresis (PHOGE).

A versatile system (PHOGE) has been developed that allows resolution of molecules of DNA megabase pair size by the use of homogeneous, orthogonal, pulsed fields. The resulting electrophoretograms have characteristics that differ from those produced by other systems for pulsed field electrophoresis. Molecules in a two-fold range of sizes can be separated with maximum resolution, or a much larger range of sizes may be separated with lower resolution but with a linear relationship of mobility to size from 50 kb, or below, to at least 1 Mb. Straight lanes and large useable gel areas, characteristic of PHOGE, are also valuable for mapping procedures or for any other circumstance in which large numbers of samples of DNA are to be directly compared. Existing models cannot explain the results obtained, because a stage of the molecular reorientation appears to result in a rate of migration greater than that occurring by reptation. We suggest a mechanism that might account for the resolution observed and also suggest that the resolution achieved by existing OFAGE-type systems may be the result of the superimposition of PHOGE and FIGE separatory mechanisms. No maximum size of molecules that may be resolved by the PHOGE system has yet been determined.     

New methods for rapid separation and detection of oligosaccharides from glycoproteins

Ion exchange chromatography at high pH with pulsed amperometric detection of the eluted glycans permitted resolution of the eight major components in the mixture of asparagine-linked glycans derived from the single glycosylation site of ovalbumin. The individual glycans were first partially separated according to size, and were characterized by fast atom bombardment-mass spectrometry and specific enzymatic degradation with beta-galactosidase and endoglycosidase H; subnanomolar quantities of all eight components could subsequently be unequivocally identified in the elution diagram. To ascertain that the chromatographic separation of the ovalbumin glycan mixture was not restricted to the asparagine-linked glycans, it was established that the corresponding mixture of reducing oligosaccharides (asparagine removed) or Asn-oligosaccharides blocked at the alpha-amino group with biotin gave very similar resolution of the eight glycans. In the absence of pure reference compounds, the quantification of the different glycans by the amperometric detection system was evaluated by comparing the electrochemical signal to the molecular ion peak intensity in the mass spectrometer. With one exception, the two methods were in good agreement, which suggests that the amperometric detection system yields a valid quantitative estimate for most of these chemically related compounds.     

Relative order determination of four Yp cosmids on metaphase and interphase chromosomes by two-color competitive in situ hybridization

Summary Two-color competitive in situ hybridization was used to cytogenetically order four Yp cosmid probes, located in the pseudo-autosomal and TDF regions. The probes were hybridized by pairs to metaphase and interphase chromosomes. On metaphase chromosomes, determination of order between sequences separated by 3 Mb from each other was possible on a statistical basis, whereas the relative position of sequences 0.6Mb apart could not be determined. On interphase chromosomes the complete order between sequences separated by 0.6– 6Mb was obtained rapidly by measuring the distances between two cosmid spots of every cosmid pair used in 28 to 60 nuclei. Results demonstrate the potential power of fluorescent in situ hybridization at interphase for high resolution cosmid mapping.     

High pressure liquid chromatographic identification of hyaluronic acid and chondroitin sulphate disaccharides.

In this report we describe a system capable of resolving all of the known unsaturated disaccharides derived from the chondroitin sulphates, dermatan sulphate and hyaluronic acid by chondroitinase digestion. This system is superior to others in that the non-sulphated and mono-, di- and tri-sulphated disaccharides can be separated with good resolution in approximately 40 min in an isocratic solvent. The system employs an amino-cyano silica gel column (Whatman Partisil 5 PAC, 25 cm) and is eluted with an isocratic solvent consisting of 48% (v/v) acetonitrile, 14% (v/v) methanol and 38% (v/v) aqueous buffer. This aqueous buffer contains 0.5 M Tris-HCl, 0.1 M boric acid, 23.4 mM sulphuric acid, pH 8.0. UV absorption is monitored at 229 nm and for most disaccharides as little as 150 ng can be reliably determined. The addition of boric acid to the eluent is essential for good resolution of all components and the addition of low concentrations of sulphuric acid is used to control the elution times of various components. The system was applied to the analysis of glycosaminoglycan standards and excellent agreement with previous compositional analyses was obtained.     

Low-molecular weight nuclear RNA components in human lymphocytes cultured without or with phytohaemagglutinin

Purified human lymphocytes were cultured without or with phytohaemagglutinin (PHA) in the presence of radioactive RNA precursors. RNA was extracted with phenol at 0°, 40° or 62°C and separated on polyacrylamide gels. RNA extracted with phenol either in presence or absence of the RNAse inhibitor diethylpyrocarbonate showed no sign of degradation when separated on 2.6 or 3% polyacrylamide gels. Ten percent gel profiles of whole cell or nuclear RNA showed a a number of small mol. wt RNA components (K, L, M, N, A, B, C, D, F) apart from tRNA, 5 S RNA and 5.5 S RNA. Profiles of cytoplasmic RNA showed only components K and L apart from tRNA, 5 S RNA and 5.5 S RNA. L, C, D and F have an electrophoretic mobility similar to the corresponding components in various ascites cells, while M, N and B may be unique for human cells.The low-molecular wt nuclear RNA components (snRNA) are found in non-stimulated as well as in PHA-stimulated cells and the relative amounts of the snRNA components are not changed during PHA-induced transformation. It is therefore concluded that the relative amounts of the different snRNA components are not related to the dynamic state of the cell.     

Structural basis and kinetics of inter- and intramolecular disulfide exchange in the redox catalyst DsbD

DsbD from Escherichia coli catalyzes the transport of electrons from cytoplasmic thioredoxin to the periplasmic disulfide isomerase DsbC. DsbD contains two periplasmically oriented domains at the N- and C-terminus (nDsbD and cDsbD) that are connected by a central transmembrane (TM) domain. Each domain contains a pair of cysteines that are essential for catalysis. Here, we show that Cys109 and Cys461 form a transient interdomain disulfide bond between nDsbD and cDsbD in the reaction cycle of DsbD. We solved the crystal structure of this catalytic intermediate at 2.85 A resolution, which revealed large relative domain movements in DsbD as a consequence of a strong overlap between the surface areas of nDsbD that interact with DsbC and cDsbD. In addition, we have measured the kinetics of all functional and nonfunctional disulfide exchange reactions between redox-active, periplasmic proteins and protein domains from the oxidative DsbA/B and the reductive DsbC/D pathway. We show that both pathways are separated by large kinetic barriers for nonfunctional disulfide exchange between components from different pathways.     

Quantification of the success of phylogenetic inference in simulations

For phylogenetic simulation studies, the accuracy of topological reconstruction obtained from different data matrices or different methods of phylogenetic inference generally needs to be quantified. Two components of performance within this context are: (1) how the inferred tree topology matches or conflicts with the correct tree topology, and (2) the branch support assigned to both correctly and incorrectly resolved clades. We present a method (averaged overall success of resolution) that incorporates both of these components. Branch support is incorporated in the averaged overall success of resolution by linearly scaling the observed support relative to that conferred by uncontradicted synapomorphies. We believe that this method represents an improvement relative to the commonly used approaches of quantifying the percentage of clades that are correctly resolved in the inferred trees or presenting the Robinson–Foulds distance between the inferred trees and the correct tree. In contrast to Bremer support, the averaged overall success of resolution may be applied equally well to distance, likelihood and parsimony analyses. © The Willi Hennig Society 2006.     

Toward plasma proteome profiling with ion mobility-mass spectrometry

Differential, functional, and mapping proteomic analyses of complex biological mixtures suffer from a lack of component resolution. Here we describe the application of ion mobility-mass spectrometry (IMS-MS) to this problem. With this approach, components that are separated by liquid chromatography are dispersed based on differences in their mobilities through a buffer gas prior to being analyzed by MS. The inclusion of the gas-phase dispersion provides more than an order of magnitude enhancement in component resolution at no cost to data acquisition time. Additionally, the mobility separation often removes high-abundance species from spectral regions containing low-abundance species, effectively increasing measurement sensitivity and dynamic range. Finally, collision-induced dissociation of all ions can be recorded in a single experimental sequence while conventional MS methods sequentially select precursors. The approach is demonstrated in a single, rapid (3.3 h) analysis of a plasma digest sample where abundant proteins have not been removed. Protein database searches have yielded 731 high confidence peptide assignments corresponding to 438 unique proteins. Results have been compiled into an initial analytical map to be used -after further augmentation and refinement- for comparative plasma profiling studies.     

Analysis of sialyllactoses in blood and urine by high-performance liquid chromatography

A sensitive and highly selective high-performance liquid chromatography (HPLC)-based method has been developed for the analysis of oligosaccharides in biological fluids. In this method, a sample of biological fluid, such as blood serum or urine, is filtered through a 10,000 molecular weight cutoff filter cartridge to remove large molecules such as proteins and lipids. The carbohydrates in the filtrate are then derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP) as described previously [Anal. Biochem. 180, 351-357, (1989)]. The derivatized carbohydrates are separated by reverse-phase HPLC and monitored by UV absorbance at 245 nm. Quantitative analysis of the carbohydrates can be achieved based on their integration values relative to a standard calibration curve. Since neutral and acidic carbohydrates can be separated by using Dowex 1-X8 anion exchange resin, this method can be used specifically to analyze neutral, acidic, and total carbohydrates in the biological fluids. Because PMP specifically reacts with reducing aldoses, interference from noncarbohydrate components present in the biological fluids is essentially eliminated. This method has proven to be highly sensitive, requiring as little as 5 pmol of analyte for reliable analysis. It has also been used successfully for pharmacokinetic analysis of carbohydrate drugs in human blood and urine samples.     

Gradient flattening of sodium dodecyl sulfate (SDS) and isoelectric focusing (IEF) gels

In addition to our previously reported versatile methods for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis [1] and isoelectric focusing [IEF]-gel [2], I have achieved molecular weight gradient flattening of the SDS-polyacrylamide gel and pH gradient flattening of the IEF gel at any segment using the same electrophoresis system. Any crowded gel segment where congregated components are not separated well can easily be widened for good separation and any dispersed gel segment where components are too far can easily be narrowed. Therefore, every gel segment can be used effectively and meaningfully because the gradient curve can be ajusted to any distribution of the components. In the crowded area, any small spots of components which could not be detected previously because of nearby heavy staining or strong radioactivity of an abundant component can be sufficiently separated from the nearby spots in a small gel without sacrificing other areas.     

Effect of different surfactants on the separation by micellar electrokinetic chromatography of a complex mixture of dipeptides in urine of prolidase-deficient patients

Prolidase deficiency is a severe disorder characterized by massive excretion of metabolites with closely related structures. At present, micellar electrokinetic chromatography is the separation method which provides the highest selectivity of structurally similar solutes. However, the structure of a surfactant can greatly affect the selectivity of separation depending on factors such as the length of hydrophobic alkyl chain or the nature of the hydrophilic group. Here we investigated the effect of three non-ionic and four anionic detergents for obtaining the best separation conditions for resolving imidodipeptide mixtures. The effect on resolution of variables such as temperature, surfactant concentrations and organic solvents was also examined. The greatest resolution was obtained at the lowest temperature studied (10°C) using 50 mM sodium borate, pH 9.3 containing 50 mM pentanesulfonate and 10% (v/v) methanol. Under these experimental conditions almost all excreted components were baseline separated and identified.