Direct vertical electroelution of protein from a PhastSystem band for mass spectrometric identification at the level of a few picomoles.

An electroelution apparatus prototype of a new design was constructed. In that design, the electric field passes vertically through the protein band located on a horizontal (PhastSystem) minigel polymerized on a net of Gel-Fix (Serva). A simple, home-made apparatus allows for electroelution of protein bands at the level of a few picomoles and their identification, after concentration, by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The technique is applicable to one-dimensional (1-D) or two-dimensional (2-D) gels of any size, but has been exemplified only by application to 1-D minigels to demonstrate the lower limits of protein load of the method. When in the course of further development of the prototype it will be combined with a modification to two dimensions of the electroelution mechanism under computer control of the high-performance gel electrophoresis apparatus (formerly of LabIntelligence), the new design appears uniquely qualified for an automated spot elution from 2-D gels under avoidance of gel sectioning.

Polydispersity of liposome preparations as a likely source of peak width in capillary zone electrophoresis.

Negatively charged liposomes consisting of phosphatidylcholine/phosphatidylglycerol/cholesterol in various ratios when subjected to capillary zone electrophoresis (CZE) in Tris-HCl (pH 8) buffer of different concentrations have been shown previously to exhibit a size-dependent migration rate at low ionic strength. The present study, focusing on the peak width under those conditions, shows that the polydispersity of liposomes correlated with, and appears to be a dominant source of, the peak width of the liposomes in CZE in a buffer of low ionic strength (2 to 5 mM Tris-HCl buffer, pH 8) at moderate electric field strengths (200 V cm(-1) or less). This finding, beyond allowing for the analysis of liposome polydispersity by CZE, suggests that the size-dependent fractionation of liposome preparations by a preparative electrophoretic technique such as free-flow electrophoresis is potentially feasible.

Sequential electroelution and mass spectroscopic identification of intact sodium dodecyl sulfate-proteins labeled with 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester.

A gel electrophoresis apparatus capable of scanning the migration path fluorometrically and of computer-directed electroelution of bands was applied to the mass spectrometric identification of sequentially electroeluted 5(6)-carboxyfluorescein-N-hydrosuccinimide ester (FLUOS)-labeled sodium dodedyl sulfate (SDS)-proteins. The masses of four electroeluted SDS-proteins under study determined by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) spectrometry are changed by 1% due to their reaction with FLUOS in a 1:5 molar ratio of protein:label, allowing for the identification of the labeled intact proteins on the basis of mass. More importantly, the partial (10 or 50%) derivatization of proteins with FLUOS does not preclude their tryptic hydrolysis, and identification of the protein on the basis of the mass spectrometric analysis of its tryptic peptides. Potentially, the procedure allows for the automated mass spectrometric identification of SDS-proteins globally labeled with FLUOS and electrophoretically separated, without need for any gel sectioning.

Stacking of unlabeled sodium dodecyl sulfate-proteins within a fluorimetrically detected moving boundary, electroelution and mass spectrometric identification.

The previously reported fluorimetric detection of sodium dodecyl sulfate (SDS)-protein in the presence of cascade blue in agarose gel electrophoresis using barbital buffer was found to be equally feasible in the absence of the fluorescent marker and using Tris-Tricinate buffer, provided that SDS was loaded with the sample but not contained in the catholyte. That fluorescent detection is thought to be due to the formation of a moving boundary between leading SDS and trailing barbital, or Tricinate buffer. This hypothesis is supported by the following evidence: (i) The fluorometrically detected band disappears with addition of SDS to the catholyte; (ii) band area is proportional to protein and/or SDS load; (iii) mobility of SDS-proteins differing in mass is the same at agarose concentrations up to 3%; (iv) lowering of protein mobility by increase in gel concentration and/or increase in the size of the SDS-protein leads to band disappearance. Fluorescent detection of the band is like to be nonspecific and due to the light scattering properties of a stack comprising moving boundaries of any analytes with net mobilities intermediate between SDS (or micellar SDS) and the trailing buffer constituent at their regulated very high concentrations. The steady-state stack of SDS-proteins in the size range of 14.4-45.0 kDa, and the transient stack of an SDS-protein of 66.2 kDa have lent themselves to electroelution and characterization by mass of the proteins after removal of SDS and buffer exchange using matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry. The possibility to form a stack of protein between leading SDS and trailing buffer anions under conditions of weak molecular sieving (open-pore gel and small-sized protein) contributes to the understanding of moving boundaries in gel electrophoresis, but in view of the narrowly defined conditions, under which this stack forms, is of limited practical significance for the gel electrophoresis of SDS-proteins.

Rapid capillary coating by epoxy-poly-(dimethylacrylamide): performance in capillary zone electrophoresis of protein and polystyrene carboxylate.

A fast and simple method for the internal coating of capillaries in capillary zone electrophoresis (CZE) is that with epoxy-poly(dimethylacrylamide) (EPDMA). Duration of coating by that method is 30 min, compared with that of 24 h when using uncross-linked polyacrylamide (PA) under otherwise identical conditions. Under the conditions used for the CZE of proteins (pH 9.0, 2% polyethylene glycol), the capillary coating with EPDMA is stable for at least 50 consecutive runs as judged by the constancy of low electroosmotic flow, equalling the stability of coating achieved by PA. Protein mobilities and protein peak asymmetry (suggestive of reversible interaction with the capillary wall) are also found to be the same in EPDMA and PA coated capillaries. Differences between EPDMA and PA coating also exist: The former is unstable upon lowering the ionic strength of the buffer to 0.003, upon the addition of sodium dodecyl sulfate (SDS) to the buffer and in application to the hydrophobic analyte, polystyrene carboxylate.

Electroelution of proteins from bands in gel electrophoresis without gel sectioning for the purpose of protein transfer into mass spectrometry: elements of a new procedure.

Electroelution of protein bands from a gel has advantages over the competitive common technique requiring gel sectioning with respect to yield, speed and the potential for computer-controlled application to multicomponent two-dimensional (2-D) gels. The electroelution design for the commercial high-performance gel electrophoresis (HPGE) apparatus represented the most advanced technique to date until the recent discontinuation of its production. The present report serves to summarize the necessary design elements for the purpose of renewing and further developing the electroelution technique. A rudimentary technique is presented by which the electroeluate is collected in a glass tube superimposed on a reversibly stained gel band and connected to an anolyte reservoir. Although the stain used is insufficiently sensitive, the technique allowed for the qualitative verification of its usefulness in the transfer of the electroeluate into mass spectrometry.

Discrimination between peak spreading in capillary zone electrophoresis of proteins due to interaction with the capillary wall and due to protein microheterogeneity.

Our study attempts to find an approach to distinguishing between the contribution to peak spreading in capillary zone electrophoresis (CZE) due to protein microheterogeneity and that due to interaction with the capillary wall, by analyzing correlations between observed peak spreading and peak asymmetry. The peak asymmetry was measured as ln[(tm-t1)/(t2-tm)] where tm, t1, and t2 are migration times at the mode of the peak and at the intersection of the peak width at half-height with the ascending and descending limbs, respectively. Two isoforms of recombinant green fluorescent protein (GFP-1 and GFP-2, 27 kDa molecular mass), glucose-6-phosphate dehydrogenase (GPD, 104 kDa), and the naturally fluorescent protein R-phycoerythrin (PHYCO, 240 kDa) were subjected to CZE in polyacrylamide-coated fused-silica capillaries of 50 and 100 microns diameters under varying conditions of protein concentration, field strength, and the initial zone length. Under conditions such that contributions to peak spreading from axial diffusion, thermal effects, and electrophoretic dispersion are negligible, the analysis of the interrelations between peak width and peak asymmetry was found to allow a conclusion as to the cause of peak spreading in CZE of protein. It appears that the peak width of GFP-2 originates mostly in protein microheterogeneity while that of GFP-1 is due to protein-capillary wall interactions. For PHYCO, both microheterogeneity and protein-capillary wall interactions contribute to peak spreading. GPD exhibits relatively little microheterogeneity or interaction with capillary walls. Thus, its peak width appears to be mostly affected by an extracolumn source of spreading such as the initial zone length.

Gel electrophoretic isolation, in the hundred microgram range, of recombinant SDS-syntaxin from sea urchin egg cortical vesicles.

Recombinant urchin syntaxin [Xa cut], electrophoresed at pH 9.0 (25 degrees C) or 10.2 (0 degrees C) in a discontinuous Tris-chloride-glycinate buffer system in the presence of 0.03% SDS in the catholyte, exhibits a multicomponent pattern in gels of a polyacrylamide concentration of 12% and 3% crosslinking. The position in the pattern of the syntaxin band was identified by reference to electropherograms of a previous study (P. Backlund, pers. comm.). The complexity of the protein composition of the preparation was reduced by selective stacking of proteins with mobilities greater than that of syntaxin. This provides a gel pattern consisting of two bands with mobilities close to that identified as syntaxin, as well as a minor, more slowly migrating, contaminant. The two major components are designated as S1 and S2, the latter being the larger species. In the absence of SDS, the preparation exhibits two pairs of protein components. Three of the proteins are charge isomers, i.e., of equal size, differing only in net charge, assumed to be forms of S1, while the fourth component is larger and is assumed to be S2. Aliquots of the preparation, containing 150 microg of protein were loaded on a cylindrical polyacrylamide gel of 18 mm diameter, and separated S1 and S2 were excised in a position defined by their characteristic values of relative mobility (Rf). Two or three gel slices, corresponding in Rf to S1 or S2, were pooled and loaded onto a Stacking Gel (5% polyacrylamide, 20% cross-linked) of 18 mm diameter, equipped with a collection chamber of 200 microL volume. The protein was electroeluted from the gel slices and concentrated into a stack by electrophoresis. The stack, marked by bromphenolblue, was allowed to migrate into the collection chamber, was collected and analyzed by protein assay and re-electrophoresis. Re-electrophoresis of S1 shows that it consists of at least three components. Recovered S1 constitutes 47% of the preparation, based on protein assay, S2 4%. S1, isolated from SDS-PAGE, exhibits an apparent Mr of 22.7 kDa, S2 one of 34.5 kDa, similar to the value of 32.6 kDa expected from the structure of syntaxin. The absence of S2 from the electroeluate re-electrophoresed at 0 degrees C and their molecular weight relationship suggest a proteolytic transformation of S2 to S1.

Recovery of sodium dodecyl sulfate-proteins from gel electrophoretic bands in a single electroelution step for mass spectrometric analysis.

Mass spectrometric analysis of proteins derived from bands in gel electrophoresis is incompatible with the covalent fluorescent labeling of the protein. Thus, if one wishes to take advantage of the capacity for computer-directed electroelution of electrophoresis apparatus with intermittent fluorescent scanning of the migration path, the protein must be labeled fluorescently in a noncovalent, reversible fashion. This was recently achieved by staining of SDS-proteins with Cascade blue and electrophoresis in barbital buffer. However, the method was not a practical one for the purpose of isolating proteins from gel electrophoretic bands and their transfer into the mass spectrometer for three reasons: (i) Ten consecutive electroelution steps were required to obviate pH changes in the electroelution chamber; (ii) electroeluates from six gel electrophoretic lanes needed to be pooled; (iii) excessive protein loads ranging from 7 to 33 microg/pool were required. The present study reports the solution to those three problems. Mass spectrometric (MALDI-TOF) characterization of five proteins was demonstrated (i) after a single electroelution step; (ii) using electroelution from a single gel of 0.3-cm(2) cross-sectional area; and (iii) using a protein load of 2 (in one case 4) microg. However, the migration rates of the Cascade blue-SDS-protein-barbital complexes derived from proteins with widely varying molecular weights proved to be the same. Thus, despite the three advances made, the method to date remains restricted to samples of single proteins.

Interactions between carbonic anhydrase and its inhibitors revealed by gel electrophoresis and circular dichroism.

Structural properties, and especially the differential stability, of complexes between carbonic anhydrase (CA) and three sulfonamide inhibitors, acetazolamide, dorzolamide and methazolamide, were investigated by spectroscopic and electrophoretic techniques. These included denaturant gradient gel electrophoresis either across a urea or a steady-state transverse sodium dodecyl sulfate (SDS) gradient. Acetazolamide, the smallest and most hydrophilic of the sulfonamides, forms the most stable complex in the presence of urea, whereas dorzolamide, with a bulky and hydrophobic structure, is most stable against the effects of SDS. At pH 7.4, complexes with dorzolamide show minimal changes in mobility across the SDS gradient, as if unaffected by the detergent, both in the presence and in the absence of excess ligand in the gel. When bound to both acetazolamide and methazolamide, on the other hand, CA displays an increase in mobility above 0.05% SDS, lower in the presence than in the absence of excess ligand. The finding of a distinct pattern for the unliganded enzyme, however, suggests the complexes can still retain the ligand, although binding of the surfactant changes their charge density. Under saturating conditions and in the presence of SDS, the surface charge of all complexes is much lower than for unliganded, denatured CA. Circular dichroism (CD) spectra clearly indicate that the increase in secondary structure and the decrease in tertiary structure brought about in CA by the presence of low concentrations of SDS are largely prevented by complexing with the inhibitors. These observations point out peculiar properties of each CA inhibitor, of potential value in the definition of their biological activities and also in the potential development of novel antagonist molecules.

Electroelution of nonfluorescent stacked proteins detected by fluorescence optics from gel electrophoretic bands for transfer into mass spectrometry.

The extreme accuracy of spectrometrically determined masses of proteins has opened the possibility to identify proteins separated as gel electrophoretic bands in the absence of specific immunologic ways of identification. For the purpose of protein transfer from gel electrophoretic bands to mass spectrometer, electroelution from the intact gel has advantages, in particular when apparatus with capacity for fluorescent scanning allows one to direct the electroelution cell over the band under computer control. To avoid fluorescent labeling of the protein which is incompatible with mass spectrometric identification, it is proposed to selectively stack the unlabeled protein and detect it by comigrating tracking dye prior to electroelution. The feasibility of the approach is exemplified in case of a single protein, but still remains to be demonstrated in conjunction with the selective stacking or unstacking of a single protein from a mixture of several proteins.

Separation efficiency in protein zone electrophoresis performed in capillaries of different diameters.

R-phycoerythrin (PHYCO, Mr 240 000), glucose-6-phosphate dehydrogenase (GPD, Mr 104 000) and two charge isomers of recombinant green fluorescent protein (GFP-1 and GFP-2, Mr 27 000) were subjected to capillary zone electrophoresis (CZE) in capillaries of 50, 100 and 150 microm inner diameter at various sample concentrations, electric field strengths, and lengths of the initial zone with the purpose of testing the hypothesis that protein - capillary wall interactions rather than thermal effects are predominantly responsible for the peak spreading of proteins in CZE. The efficiency of CZE was expressed in terms of the number of theoretical plates, N, or the plate height corrected by subtracting the contribution from initial zone length, H'. The latter has the advantage of solely reflecting contributions to the separation efficiency arising from intracolumn peak spreading in capillaries of different diameters. The separation efficiency measured varied widely, by two orders of magnitude, for these proteins under identical conditions, with GPD exhibiting the highest and PHYCO the lowest values of N. H' was found to be independent of sample concentrations within the concentration ranges studied, 1-100 microg/mL for PHYCO and 100-1000 microg/mL for GPD, while exhibiting a decrease with sample concentration for GFP, especially in 150 microm diameter capillaries, within the concentration range 1-100 microg/mL. H'was also found to be independent of electric field strength up to 300-400 V/cm for PHYCO and GFP. In all experiments, the CZE of proteins in 100 microm diameter capillaries provided a higher or, at least, equal efficiency, compared to that in 50 or 150 microm diameter capillaries. It may be concluded that the protein - capillary wall interactions and protein microheterogeneity are the dominant sources of peak spreading and their specific combinations are thought to be responsible for the wide variation in separation efficiency between proteins in CZE observed under identical conditions.

Gel electrophoretic distinction between Congo Red nonreactive beta-amyloid (1-42) and beta-amyloid (1-40).

Congo-Red nonreactive beta-amyloid (1-42) exhibits in gel electrophoresis (pH 8.82, 0.01 M ionic strength, 2 degree C) a surface charge density larger than that of the corresponding peptide of length (1-40), and a size indistinguishable from that of (1-40).

Size-dependent retardation and resolution by electrophoresis of rigid, submicron-sized particles, using buffered solutions in presence of polymers: a review of recent work from the authors' laboratory.

Capillary zone electrophoresis (CZE) was conducted in buffered solutions of polyacrylamide (PA) and polyethylene glycol (PEG) to find the degree and the manner in which separation and resolution of submicron-sized rigid spherical polystyrene sulfate and carboxylate particles were affected by the presence of those polymers. In resolving pairs of representative particles, maximal resolution was observed at or near the entanglement threshold concentration, c*, of the polymer. The value of that maximum represents a several-fold increase in resolution. Since c* can be calculated from intrinsic viscosity, and the latter from the molecular weight of the polymer (and some constants available in the literature), optimally resolving polymer conditions become predictable. The maximum can also be experimentally determined by measuring intrinsic viscosity and calculating c*, or by either systematically varying the concentration of a polymer of constant molecular weight or by varying the molecular weight of a polymer at constant concentration. An optimally resolving field strength is superimposed on the maximally resolving condition of polymer concentration and weight.

Capillary zone electrophoresis of sub-microm-sized particles in electrolyte solutions of various ionic strengths: size-dependent electrophoretic migration and separation efficiency.

To gain insight into the mechanisms of size-dependent separation of microparticles in capillary zone electrophoresis (CZE), sulfated polystyrene latex microspheres of 139, 189, 268, and 381 nm radius were subjected to CZE in Tris-borate buffers of various ionic strengths ranging from 0.0003 to 0.005, at electric field strengths of 100-500 V cm(-1). Size-dependent electrophoretic migration of polystyrene particles in CZE was shown to be an explicit function of kappaR, where kappa(-1) and rare the thickness of electric double layer (which can be derived from the ionic strength of the buffer) and particle radius, respectively. Particle mobility depends on kappaR in a manner consistent with that expected from the Overbeek-Booth electrokinetic theory, though a charged hairy layer on the surface of polystyrene latex particles complicates the quantitative prediction and optimization of size-dependent separation of such particles in CZE. However, the Overbeek-Booth theory remains a useful general guide for size-dependent separation of microparticles in CZE. In accordance with it, it could be shown that, for a given pair of polystyrene particles of different sizes, there exists an ionic strength which provides the optimal separation selectivity. Peak spreading was promoted by both an increasing electric field strength and a decreasing ionic strength. When the capillary is efficiently thermostated, the electrophoretic heterogeneity of polystyrene microspheres appears to be the major contributor to peak spreading. Yet, at both elevated electric field strengths (500 V/cm) and the highest ionic strength used (0.005), thermal effects in a capillary appear to contribute significantly to peak spreading or can even dominate it.

Size-dependent electrophoretic migration and separation of liposomes by capillary zone electrophoresis in electrolyte solutions of various ionic strengths.

The size-dependent electrophoretic migration and separation of liposomes was demonstrated and studied in capillary zone electrophoresis (CZE). The liposomes were extruded and nonextruded preparations consisting of phosphatidylcholine/phosphatidylglycerol/cholesterol in various ratios and ranging from 125 to 488 nm in mean diameter. When liposomes of identical surface charge density were subjected to CZE in Tris-HCl (pH 8) buffers of various ionic strengths (0.001-0.027), they migrated in order of their size. Size-dependent electrophoretic migration and separation of liposomes in CZE can be enhanced or brought about by decreasing the ionic strength of the buffer. It was shown that size-dependent migration is primarily a function of kappaR, where kappa(-1) is the thickness of the electric double layer (which can be derived from the ionic strength, I, of the buffer) and R, the liposome radius. Liposome mobility depends on kappaR and surface charge density in a manner consistent with that expected from the Overbeek-Booth electrokinetic theory. Thus, the relaxation effect appears to be the physical mechanism underlying the size-dependent electrophoretic separation of liposomes.

Capillary zone electrophoresis of proteins in semidilute polymer solutions: inter- and intra-polymer predictability of size-dependent retardation.

The retardation of three "spherical" proteins with Stokes' radii of 2.0, 2.4, and 3.0 nm (35-104 kDa) was studied in capillary zone electrophoresis (CZE), using semidilute solutions of polyethylene glycol (PEG), linear polyacrylamide (PA), and polyvinyl alcohol (PVA). The purpose was to test the models predicting that the ratio of particle radius, R, to the mesh size of polymer network (the correlation or screening length of a semidilute polymer solution), xi, directly governs the size-dependent retardation in the form: mu/muo = exp (-R/xi). Here xi = kc-0.75, where c is polymer concentration and the numerical factor kcan be calculated based on polymer molecular weight. In application to polymers in a "good solvent" (PA and PEG in the aqueous buffer) and to proteins of 2.4 and 3.0 nm radius, that relation between relative mobility and R/xi was found to be obeyed for PA, while for PEG the value of k derived from retardation experiments significantly exceeded that which was theoretically calculated. Thus, the retardation appears to be polymer-specific, rather than universal, even for polymers in a "good solvent". It is suggested that, in that case, retardation of proteins of R > 2 nm be quantitatively described in the form mu/muo = exp[-p(R/xi], where p is a parameter depending on monomer type and/or polymer polydispersity. For PVA, the logarithm of mu/muo was found to be linearly related to c (in line with the prediction that the aqueous buffer is a "poor solvent" for this polymer) and to be near-independent of R.

Gel electrophoretic distinction between toxic and nontoxic forms of beta-amyloid (1-40).

The in vitro toxicity of synthetic beta-amyloid (1-40) correlates with its binding to Congo red (CR). Potentially, therefore, CR binding to the beta-amyloid containing neuritic plaques in Alzheimer's disease could be used diagnostically. Using polyacrylamide under nondenaturing conditions, the present study shows that both CR binding and nonbinding synthetic beta-amyloid exhibits multiple charge-isomeric and size-isomeric species. The CR binding species exhibit values of free electrophoretic mobility, related to the surface charge density of the protein, which are less than those of the CR non-binding species within 95% confidence limits. Since surface net charge and solubility are correlated, the decreased solubility of the CR binding species may be responsible for the relative abundance and CR binding of beta-amyloid in the neuritic plaques of Alzheimer patients.