N-terminal and internal sequence determination of microgram amounts of proteins separated by isoelectric focusing in immobilized pH gradients

Isolation of microgram amounts of proteins and submicrogram quantities of peptides in a form suitable for sequence analysis is a key step in high sensitivity protein sequencing technology. Recently, methods have been developed which allow the direct, high yield, recovery of microgram amounts of sequenceable proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or two-dimensional gel electrophoresis. In the present publication, we describe an extension of these methods to obtain N-terminal or internal sequence information from proteins separated by isoelectric focusing in polyacrylamide gels containing immobilized pH gradients. For N-terminal sequence analysis, separated proteins were electrophoretically transferred (electroblotted) onto chemically-modified glass fiber sheets, a support compatible with Edman degradation chemistry. Transferred protein bands were detected on the support, cut out and directly inserted into the cartridge of a gas-phase protein sequenator. For internal sequence analysis, separated proteins were electrophoretically transferred onto nitrocellulose. Protein bands were detected by staining, cut out and the proteins subjected to enzymatic digestion directly on the support. The resulting cleavage fragments (peptides) were released into the supernatant where they were recovered and separated by narrow-bore reversed-phase high performance liquid chromatography for sequence analysis. The potential of this methodology is illustrated by the comparative peptide mapping of isoforms of bovine carbonic anhydrase.     

A simple method for determining principal-stress directions in embedded-polariscope models

A disadvantage of the embedded-polariscope method is the inability to observe isoclinics because the embedded Polaroid sheets are fixed within the model. Perforating the embedded active layer with an array of small-diameter holes allows observation of stress trajectories in the layer from the isochromatic patterns. Further, it is shown that the full-field fringes are little disturbed by the presence of the small perforations. The techniques of model fabrication are described, as well as an extension of the method to reveal bending-stress trajectories in plates loaded out of plane.     

Strain-optic law for a certain class of birefringent composites

A strain-optic law for a certain class of birefringent fiber-reinforced composite materials is developed. The strain-optic law requires both mechanical and optical characterization of the material. Previous investigations have hypothesized the existence of three independent optical properties for the anisotropic birefringent material. The strain-optic relation developed in this study, however, requires only a single strain-optic coefficient, coupled with four independent mechanical material properties, for prediction of the optical response of a birefringent anisotropic material. Experimental results are presented which show good agreement with the developed strain-optic relation. Finally, the isoclinic parameter is investigated and preliminary evidence is presented which indicates a correspondence between principal-strain direction and optical isoclinic angle.     

Experimental Mechanics of Fiber Reinforced Composite Materials

Experimental Techniques -     

On the birefringent-coating method of stress analysis for fiber-reinforced laminated composites

A general relation based upon laminated-plate theory is developed which relates the state of stress throughout the thickness of the fiber-reinforced composite laminate to experimentally determined surface strains. The usefulness of the developed relation is discussed through an example given for a typical graphite-epoxy structural laminate. Birefringent coatings employed as a method to determine laminate surface strains are investigated. Isochromatic disturbances in the vicinity of a laminate free-edge, due to a Poisson's ratio mismatch between coating and laminate and interlaminar stress transfer between discrete lamina of the laminate, are investigated through exact elasticity solutions.     

Reversed-phase liquid chromatography of immunoglobulin G molecules and their fragments with the diphenyl column

A diphenyl column was able to resolve two closely related monoclonal IgG2 molecules, while a C8 column failed to separate these IgGs under identical chromatographic conditions. The diphenyl column also showed a better separation of a mixture of two light and two heavy chains than the C8 column. The influence of amino acid side chains from protein sequences in binding to the diphenyl and C8 stationary phases was studied by using a set of synthetic peptides with the sequence GXXLLLKK, where X represents substitution with all of the 20 amino acids. Peptides containing aromatic amino acids showed a greater binding on the diphenyl column than on the C8 column. This increase in retention was attributed to pi-pi interactions between the aromatic amino acid side chains and the diphenyl ligand. Based on the retention of peptides on the diphenyl column, new retention coefficients were assigned for the separation of proteins. A good correlation was observed between the sum of retention coefficients (SigmaRc) for IgGs and their retention time on the diphenyl column. On-column hydrogen-deuterium exchange showed that the diphenyl column had a larger surface of interaction with protein than the C8 column. pi-pi interactions and the large contact surface resulted in improved resolution of IgGs and their fragments on the diphenyl column.     

Reversed-phase liquid chromatography/mass spectrometry analysis of reduced monoclonal antibodies in pharmaceutics

A reversed-phase LC/MS method was developed for reduced antibodies that provides efficient separation of light chain and two variants of heavy chain containing N-terminal glutamine and pyroglutamic acid. The best separation was achieved on Zorbax CN and Varian Pursuit DiPhenyl columns eluted with increasing percentage of n-propanol and acetonitrile in 0.1% trifluoroacetic acid. Although glutamine was genetically coded for the N-terminal residue of heavy chain of a monoclonal antibody used in this study, we found that most of it (70%) was converted to pyroglutamate during production. The conversion process continued in vitro and was monitored by the method. Deconvoluted electrospray ionization mass spectrum of the heavy chain revealed the glycosylation profile of a single N-linked sugar including a-, mono-, and di-galactosylated biantennary glycans and a 5-mannose sugar form.     

Comparison of LC and LC/MS methods for quantifying <Emphasis Type="Italic">N</Emphasis>-glycosylation in recombinant IgGs

High-performance liquid chromatography (LC) and liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-MS) methods with various sample preparation schemes were compared for their ability to identify and quantify glycoforms in two different production lots of a recombinant monoclonal IgG1 antibody. IgG1s contain a conserved N-glycosylation site in the fragment crystallizable (Fc) subunit. Six methods were compared: (1) LC/ESI-MS analysis of intact IgG, (2) LC/ESI-MS analysis of the Fc fragment produced by limited proteolysis with Lys-C, (3) LC/ESI-MS analysis of the IgG heavy chain produced by reduction, (4) LC/ESI-MS analysis of Fc/2 fragment produced by limited proteolysis and reduction, (5) LC/MS analysis of the glycosylated tryptic fragment (293EEQYNSTYR301) using extracted ion chromatograms, and (6) normal phase HPLC analysis of N-glycans cleaved from the IgG using PNGase F. The results suggest that MS quantitation based on the analysis of Fc/2 (4) is accurate and gives results that are comparable to normal phase HPLC analysis of N-glycans (6).     

Rheological and dynamic thermomechanical propert ies of epoxy composites reinforced with single- and multiwalled carbon nanotubes

An epoxy resin (Epon 828) was filled with single- and multiwalled carbon nanotubes (SWCNT and MWCNT) in two steps by using the high shear mixing and ultrasonication techniques. The melt flow of the composites was characterized in a plate/plate rheometer. The thermomechanical properties of the composites were determined in dynamic mechanical analysis tests performed at various frequencies and temperatures. It was found that the incorporation of SWCNT or MWCNT increased the viscosity and stiffness of epoxy above its glass-transition temperature. The time-temperature superposition principle was employed to estimate the storage modulus of the composites as a function of frequency (f = 10^–33–10³ Hz) in the form of master curves.     

Constriction of a microwave-induced plasma by a magnetic pinch

The microwave-induced plasma has been widely studied as a spectrochemical excitation source. The plasma is generally maintained in a quartz tube, and energy coupled via a cavity or antenna. Previous work has shown the importance of the electron density; plasma performance is improved as the instrumental parameters are adjusted to increase the electron density. Another method to increase the electron density is to constrict the plasma. Additionally, energy losses via wall-collision would be decreased if the plasma were moved away from the walls. This communication presents such a study, in which the plasma is ‘pinched’ by the application of an external magnetic field. Preliminary results show an increase in intensity by a factor of two. The constriction also tends to improve the atomization processes. Measurements performed on plasmas which contain carbon monoxide show a greater increase in the carbon atomic emission than the carbon monoxide molecular emission.