Electrospray mass spectra from protein electroeluted from sodium dodecylsulfate polyacrylamide gel electrophoresis gels

Electrospray ionization/tandem mass spectrometry of proteins separated on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels is severely limited by the requirement that the protein be completely separated from the SDS. As shown here, the gaseous noncovalent SDS adducts of protonated proteins thus formed can be dissociated by infrared irradiation. ESI spectra from myoglobin in SDS-containing solutions show molecular ions adducted with up to 15 dodecyl sulfates, but ir irradiation of these ions causes complete dissociation to expel the SDS.     

Characterization of sodium dodecylsulfate polyacrylamide gel electrophoresis-separated M. agalactiae membrane antigens by mass spectrometry

Mycoplasma membrane proteins are generally designated according to their apparent molecular weight measured by SDS-PAGE. Several results about mycoplasma membrane antigens are conflicting because some doubts are emerging about the accuracy of the method utilised to identify the antigens. Aim of this work, was to characterise proteins separated after sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE)-mass spectrometry to allow an uncontroversial designation of the antigens. Fifteen proteins with molecular weights ranging from 15,000 to 80,000 Da had been excised from gel and their whole molecular weight and proteolytic pattern had been determined using MALDI-TOF. The peptide pattern obtained using trypsin digestion allowed us to identify LipA, P48, P59, P80 and P40. Some other proteins showed analogies to proteins of Mycoplasma genitalium or Mycoplasma pneumoniae the only Mycoplasmas completely sequenced. There wasn't a close correspondence between the SDS-PAGE apparent molecular weight (generally used to name the proteins), the gene derived calculated mass and the molecular weight of whole proteins measured by MALDI-TOF. Only micro sequence data obtained by MS/MS allowed us to identify LipC, described as one of the most important Mycoplasma agalactiae antigens. This protein was found in correspondence with the 50 kDa region, instead of the 25 kDa region, confirming a phenomenon that we previously described.     

Coarse-grained molecular dynamics simulation of self-assembly of polyacrylamide and sodium dodecylsulfate in aqueous solution

Coarse-grained molecular dynamics simulations have been performed to study the self-assembly of polymer, polyacrylamide (PAM) and surfactant, sodium dodecylsulfate (SDS) in aqueous solution. Our simulations revealed that PAM curled into clusters in the absence of SDS, while it was stretched if SDS was added. For the SDS-PAM complexes, the aggregate formation process can be divided into three stages: firstly, PAM quickly absorbs some SDS monomers until the radius of gyration (Rg) of polymer reaches a minimum; then, PAM stretches and the Rg of PAM increases due to more and more adsorbed SDS; ultimately, the commonly accepted "necklace" structure is formed with PAM located at the interface of the hydrophobic and hydrophilic regions of the SDS micelle. The main driving force for the association was hydrophobic interactions between the polymer backbone and the surfactant hydrophobic tails. As the concentration of SDS increased, the Rg of PAM increased up to a maximum, indicating the polymer was saturated with surfactant.     

Electrophoretic investigation of the seed proteins of the cotton plantGossypium hirsutum

The proteins of the buffer-soluble fraction of the seeds of cotton plants of the speciesGossypium hirsutum, wild and ruderal varieties, have been investigated by electrophoresis in polyacrylamide gel and have been subdivided into two classes. Characteristic components have been detected in the protein spectra of these samples. In diploid species of the cotton plant, in addition to characteristic protein components, individual components are also observed.Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 736–738, September–October, 1995. Original article submitted November 21, 1994.     

Electrophoretic heterogeneity of maize histones

Histones from maize embryos and seedlings have been isolated using a fast extraction procedure. Three different electrophoretic systems have been applied for the study of the heterogeneity of maize core histones. Electrophoresis in acetic acid/urea polyacrylamide gels, containing high concentrations of urea, resulted in optimum fractionation of the core histones and especially of histone H4. Sodium dodecyl sulfate-containing polyacrylamide gels were not useful for the fractionation of maize histone classes H2a and H2b, nor for the various subfractions of H3 and H4. Gels containing Triton X-100, used for the dimension in two-dimensional electrophoresis proved to be efficient for the separation of all histone classes, as well as their structural variants and chemical modifications. Maize core histones have been oxidized in an attempt to define which of the Triton X-100 resolved subfractions represent oxidation forms.     

A comparative study of the histones of some varieties of cotton plant

Tetraploid varieties of the cotton plant belonging to the medium- and thin-fibered types have been studied with respect to their histone/DNA ratio, their content of histone fractions, and the amino acid composition of the total histones. It has been shown that these varieties differ with respect to their contents of the lysine-rich histone fraction Hl, and also by the amounts of certain amino acids in the total histone.V. I. Nikitin Institute of Chemistry, Academy of Sciences of the TadzhSSR, Dushanbe. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 99–102, January–February, 1985.     

Supramolecular chirality of sodium dodecylsulfate

The aggregative chirality of sodium dodecylsulfate has been discovered as a new kind of supra-molecular chirality.     

Ultrafiltration of sodium dodecylsulfate solutions

The present paper is devoted to ultrafiltration (UF) of aqueous solutions containing micellized sodium dodecylsulfate (NaDS) through zircon membranes, following permeation data of cetyltrimethylammonium bromide (CTABr), which we have reported recently.The experimental results are analyzed using the same model of diffusion in the membrane matrix based on the thermodynamics of irreversible processes (TIP). A comparison with the classical model of resistances in series leads to a new analysis of the measured resistances in terms of membrane–permeant and permeant–permeant interactions. NaDS micelles being able to cross the membrane as permeants, in contrast with CTABr micelles, the comparison of the behaviors of anionic and cationic surfactants is worthwhile. The resistance of the membrane strongly depends on the composition of the feed. The TIP approach allows us to relate the membrane resistance to the concentrations of all the species present in the feed, namely free ions, spherical micelles and rod-like aggregates. It is shown that the resistance is highly influenced by free ions (dodecylsulfate (DS⁻) and sodium (Na⁺)), very slightly by negatively charged spherical micelles, and weakly by rod-like aggregates, also negatively charged.     

Rapid analysis of covalently and non-covalently fluorophore-labeled proteins using ultra-thin-layer sodium dodecylsulfate gel electrophoresis

Gel electrophoresis is one of the most frequently used tools for the separation of complex biopolymer mixtures. In recent years, there has been considerable activity in the separation and characterization of protein molecules by sodium dodecylsulfate (SDS) gel electrophoresis with particular interest in using this technique to separate on the basis of size and to estimate molecular mass and protein purity. Although the method is informative, it is cumbersome, time consuming and lacks automation. In this paper we report an automated, high-performance SDS gel electrophoresis system that is based on electric-field-mediated separation of SDS-protein complexes using an ultra-thin-layer platform. The integrated fiber optic bundle-based scanning laser-induced fluorescence detection technology readily provided high sensitivity, real-time detection of the migrating solute molecules. Rapid separations of covalently and non-covalently labeled proteins were demonstrated in the molecular mass range 14,000 to 205,000 in less than 9 and 16 min, respectively. Excellent quantitation and lane-to-lane migration time reproducibility were found for all the solute components using the multilane separation platform. The limit of detection was found to be 1.5-3 ng/band for both labeling methods, with excellent linearity over a six times serial double-dilution range. Molecular mass calibration plots were compared for both covalently and non-covalently labeled proteins. A linear relationship was found between the molecular mass and electrophoretic mobility in the case of covalently labeled samples, while a non-linear relationship was revealed for the non-covalently labeled samples.     

Adsorption of sodium dodecylsulfate on chrysotile

Adsorption of sodium dodecylsulfate (SDS) onto chrysotile from aqueous solutions was investigated along with varying temperature, ionic strength and surface treatments. Commercial chrysotile fibers were treated by sonication or extensive washings. The ratio of adsorbed SDS per gram of chrysotile is approximately constant with varying chrysotile masses. A steady state is reached after about 2 h of contact between SDS and chrysotile. In general, less surfactant is adsorbed on the sonicated chrysotile than on the extensively washed chrysotile. For the sonicated chrysotile, isotherms presented an adsorption maximum in the region of the surfactant critical micelle concentration, when the experiments were carried out without ionic strength control. The adsorption maximum is due to the presence of magnesium ions in the solution, which can form complexes with dodecylsulfate ions. For the extensively washed chrysotile, the isotherm behavior is similar to that obtained with sonicated chrysotile in the presence of an inert electrolyte. No significant difference in adsorption of SDS on the extensively washed chrysotile was observed when varying temperature or ionic strength. The adsorption of SDS was found to be dependent on the prior surface treatment.     

Observation of sodium gel-induced protein modifications in dodecylsulfate polyacrylamide gel electrophoresis and its implications for accurate molecular weight determination of gel-separated proteins by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry (TOFMS) can potentially provide accurate molecular weight information of proteins separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Several issues related to resolution and accuracy of molecular weight measurement are investigated by using a time-lag focusing MALDI-TOF mass spectrometer. The effects of the gel components SDS, glycerol, and tris buffer on the mass spectral signals are studied systematically. Glycerol and tris buffer are shown to have little or no effect on resolution and mass accuracy, whereas SDS degrades sensitivity, resolution, and mass accuracy even at low concentrations. A simple and fast gel extraction technique is presented which is capable of detecting proteins loaded at the low-picomole level on the gel. The sample preparation procedure used in this work appears to remove most of SDS from the gel, thereby reducing the peak broadening effect caused by SDS and resulting in high resolution and accurate measurement of proteins. However, for proteins containing cysteines, the molecular ions are composed of a distribution of acrylamide-protein adducts likely formed by reaction with unpolymerized acrylamide in the gel during the gel separation process. The implications of gel-induced protein modifications on the accurate molecular weight measurement of gel-separated proteins are discussed.     

Novel cross-linked polyacrylamide matrices: an investigation using gradient gel electrophoresis

Gradient gel electrophoresis was used to examine the separation properties of novel cross-linking compounds for polyacrylamide (PAAm). At low %T and at the same %C protein migration difference is accentuated for bismethacrylamide cross-linked networks relative to bisacrylamide cross-linked networks. Similar properties were observed for cyclic monomers at low %T. This trend is maintained throughout the gradient. However, at higher %T migration differential relative to N,N'-methylenebisacrylamide (Bis) was less pronounced. Evidence from gradient gels suggests that reactivity and functionality of vinyl groups impose an overriding control over network formation.     

Modification of the immobilized metal affinity electrophoresis using sodium dodecyl sulfate polyacrylamide gel electrophoresis

Modification to the original immobilized metal affinity electrophoresis (IMAEP) technique is presented. SDS-PAGE is used instead of native PAGE for improved extraction of phosphoproteins from a mixture of proteins. Protein samples treated with 2% w/v SDS instead of native sample buffer ensure that proteins are negatively charged. These negative charges on the proteins assure that the proteins migrate electrophoretically towards the anode regardless of their pI values and hence pass through the region embedded with the metal ions. Another benefit of treating proteins with SDS is that it unfolds the phosphoproteins exposing the phosphate groups to facilitate the metal-phosphate interactions. Phosphorylated ovalbumin can only be extracted after SDS sample buffer treatment. Data show that there is no detrimental effect upon SDS treatment on the extraction of phosphoproteins from a mixture of proteins. Electrophoretic migration of phosphoproteins ceases upon encounter with metal ions like Al+3, Ti+3, Fe+3, Fe+2, and Mn+2 whereas non-phosphorylated proteins migrate freely.     

Iron-regulated proteins in Phanerochaete chrysosporium and Lentinula edodes: differential analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional polyacrylamide gel electrophoresis profiles

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis (2-DE) were used to identify iron-responsive proteins in the white-rot species (Phanerochaete chrysosporium and Lentinula edodes), by comparing the differential patterns of cellular and membrane proteins obtained from iron-sufficient and iron-deficient mycelia. Six cellular proteins induced by iron restriction have been observed in SDS-PAGE for P. chrysosporium and twelve for L. edodes. In 2-DE, the numbers of iron-restricted induced proteins were 12 and 9, respectively, in a resolution range of 15-60 kDa and pI 4.5-8.1. SDS-PAGE for the plasma membrane protein did not show differences, whereas the outer-membrane protein profile showed 6 and 5 proteins induced by iron depletion in P. chrysosporium and L. edodes, respectively. The results presented here are important data to unravel mechanisms of biosynthesis and/or transport of the iron-complexing agents in ligninolytic fungi and to further correlate them to the ligninolytic processes.     

Reduction in the antigen-binding in polyacrylamide gel

Under conditions of antibody excess, antibodies that had been immobilized by physical entrapment in a polyacrylamide gel matrix consistently removed less radiolabeled microprotein antigen from solution than did the same antibodies chemically conjugated to Sepharose beads. This reaction is believed to relate to the difficulty of formation of secondary antibody-antigen lattices when the molecules are held in “cells” in the polymer matrix.     

Electrophoretic transfer of proteins across polyacrylamide membranes

The electrophoretic transfer of purified proteins has been examined in a Gradiflow "Babyflow BF100" unit. A number of factors affect protein separation within this preparative electrophoresis system. We established that the rate of protein transfer was proportional to the applied voltage. The transfer is slowest at the isoelectric point (pI) and increased the further away the pH was from the pI of the protein. Protein transfer was found to be independent of the ionic strength of the buffer, for buffers that excluded the addition of strong acids or strong bases or sodium chloride. Transfer decreased as the pore size of the membrane decreased. Finally, transfer was inhibited at high salt concentrations in the protein solution, but remained unaffected when urea and non-ionic detergents were added to the solution. To increase the speed of protein separations, buffers with low conductivity should be used. A pH for the optimal separation should be selected on the basis of the relative pI and size of the target proteins and that of the major contaminants.