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Polyacrymalide gel isoelectric focusing (PAGIF) in thin-layer was used to resolve proteins of Mycoplasma spp., Acholeplasma spp. and of eight strains of Ureaplasma urealyticum (T-Strain). A mixture of urea, Triton X-100, and dithioerythritol was used to solubilize sonically disrupted cells.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 127-134).

The work presented in this dissertation centers around the development of analytical tools for the study of advanced proteomics. Section 1 of this work reviews the need for high efficiency protein separation techniques. Dynamic isoelectric focusing (DIEF) is new technique similar to capillary isoelectric focusing (CIEF) invented by Dr. Luke Tolley at Southern Illinois University Carbondale. Using DIEF, the electric field inside the separation capillary can be modified using high voltage electrodes, additional to the anode and cathode, to control the depth and shape of the resulting pH gradient. By changing the pH gradient, the location and width of focused protein bands can be controlled. As a new analytical technique, the development of DIEF required the design and fabrication of special holders which allow for electrical connections to be made at lengths along the separation capillary. These holders were also designed to have a removable section of capillary to extract very specific pH range proteins from high-resolution separations. Higher throughput DIEF systems were investigated, as well as multiplexed DIEF systems. Section 2 covers the topic of dynamic isoelectric/anisotropy ligand binding assay (DIABLA). DIABLA is a new method used to identify proteins in a complex sample that bind to a known molecule. DIABLA has the potential to be used in two complimentary ways, discovery mode and scanning mode. Both modes are accomplished by using DIEF, followed by fluorescence anisotropy as a sensitive detection method. This allows the entire length of capillary to be scanned to identify areas of non-zero anisotropy, which indicate binding interactions between the protein and target molecule. The binding protein(s) can then be extracted using the removable section of capillary from the DIEF holder, and can be identified by using a second dimension analysis, such as LC/MS/MS. DIABLA was verified in a series of proof-of-concept experiments in both discovery and scanning modes. These experiments involved fluorescently tagging proteins that were focused in the presence of a ligand tagged with a different fluorophore. The usefulness of DIABLA as a separation technique was demonstrated in four specific analyses of complex protein samples in Chapter 10.

The glycosylation features of CSF oligoclonal IgG, and possible changes in N-glycans of CSF IgG in multiple sclerosis (MS) were studied. After isoelectric focusing (IEF) of CSF, bands were detected using biotinylated lectins and avidin-horseradish peroxidase. Concanavalin A (Con A) binding showed that mannose exists throughout the pH range of oligoclonal IgG. Sambucus nigra antigen (SNA) bound acidic and neutral oligoclonal IgG only, suggesting that alkaline oligoclonal IgG is deficient in sialic acid. Deglycosylation of CSF IgG using peptide-N-glycosidase F suggested that the range of isoelectric points of oligoclonal IgG bands is not due to carbohydrate differences alone. Lectin immunoassays, whereby protein A purified IgG was captured by anti-IgG coated tubes and probed using a range of biotinylated lectins, were used to compare 13 CSF samples from MS patients with 14 control samples. With Con A binding, a significantly higher mean and larger variance was found for the MS group (t-test: P < 0.05). Con A binding correlated with CSF [IgG]/[total protein]% (r=0.390; P=0.0443). Using HPLC to separate oligosaccharides released from IgG by hydrazinolysis and labelled with 2-aminobenzamide, glycans were determined in 7 CSF samples with oligoclonal IgG, and 6 CSF samples without. The ratio of the peak for biantennary fucosylated agalactosyl glycans to total monogalactosylated glycan peaks was lower for the oligoclonal IgG samples (t-test: P=0.0141). The overall results suggested that glycosylation changes occur in CSF IgG in MS, and that oligoclonal IgG contains less sialic acid but more galactose than polyclonal IgG.

Thesis (Ph. D.)--Washington State University, December 2007. / Includes bibliographical references.

abstract: Bioanalytes such as protein, cells, and viruses provide vital information but are inherently challenging to measure with selective and sensitive detection. Gradient separation technologies can provide solutions to these challenges by enabling the selective isolation and pre-concentration of bioanalytes for improved detection and monitoring. Some fundamental aspects of two of these techniques, isoelectric focusing and dielectrophoresis, are examined and novel developments are presented. A reproducible and automatable method for coupling capillary isoelectric focusing (cIEF) and matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) based on syringe pump mobilization is found. Results show high resolution is maintained during mobilization and &beta-lactoglobulin; protein isoforms differing by two amino acids are resolved. Subsequently, the instrumental advantages of this approach are utilized to clarify the microheterogeneity of serum amyloid P component. Comprehensive, quantitative results support a relatively uniform glycoprotein model, contrary to inconsistent and equivocal observations in several gel isoelectric focusing studies. Fundamental studies of MALDI-MS on novel superhydrophobic substrates yield unique insights towards an optimal interface between cIEF and MALDI-MS. Finally, the fundamentals of isoelectric focusing in an open drop are explored. Findings suggest this could be a robust sample preparation technique for droplet-based microfluidic systems. Fundamental advancements in dielectrophoresis are also presented. Microfluidic channels for dielectrophoretic mobility characterization are designed which enable particle standardization, new insights to be deduced, and future devices to be intelligently designed. Dielectrophoretic mobilities are obtained for 1 µm polystyrene particles and red blood cells under select conditions. Employing velocimetry techniques allows models of particle motion to be improved which in turn improves the experimental methodology. Together this work contributes a quantitative framework which improves dielectrophoretic particle separation and analysis. / Dissertation/Thesis / Ph.D. Chemistry 2011

Doctor of Philosophy / Department of Chemistry / Christopher T. Culbertson / In research and industry, almost all chemical analysis methods involve the separation and detection of compounds. Typically, these separations are performed using traditional methods that require volumes in the 10 μL to 10 mL range of sample and in the 200 mL to 2 L range for solvents. These methods are not suitable for low-concentration, volume-limited samples frequently associated with biochemical studies. One way to overcome these limitations is to move the separation and detection to the microscale. The use of the microscale separation technologies enables the study of biological systems that have, until now, been out of reach due to their small volumes or low concentrations. The research presented in this dissertation will discuss two examples of this shift to microscale separation technologies which can solve some small volume sample challenges. These include the detection of protease activity in blood samples for use in cancer detection and the identification of immune system cascade proteins in the mosquito Anopheles gambiae. In Chapter 2 a microfluidic method and device is proposed to monitor protease activities for cancer detection. In this method nanobiosensors are used to measure enzyme activity in biological fluids. These nanobiosensors consist of iron-iron oxide magnetic nanoparticles that are attached to peptide substrates specific for proteases through a disulfide bond. The nanobiosensors are controlled using a neodymium magnet which is attached through a 3D printed adaptor to a rotating motor for mixing and a linear stage to move the nanoparticles between different sections of the device. The separation and detection sections of the device are explained in Chapter 3. Chapter 3 describes the fabrication and optimization of a simple device for microfluidic isoelectric focusing(IEF). IEF is a separation method in which analytes are separated based upon their isoelectric, i.e. neutral charge, points. A reducing agent can be added to the IEF buffer to detach the nanoparticle from the peptide substrate, releasing it for focusing. IEF is also a concentration as well as separation method that will allow the peptide substrates to be focused up to 10⁶ fold. It has a high peak capacity and produces reliable, reproducible separation patterns based on the isoelectric point of the peptide. To meet the detection limits required for cancer detection with proteases, scanning laser induced fluorescence is selected as the method of detection. This scanning system can monitor the separation over time to observe the parameters affecting the separation which cannot be done with typical point or imaging detection systems and allows better separation. This custom automatic detection system can distinguish focused samples of 500 fM from the background with minimal noise from the scanning system. In Chapter 4 the identification of serine protease and inhibitor binding complexes in A. gambiae hemolymph using magnetic bead immunoaffinity chromatography was attempted. These proteases play a key role in the insect innate immunity system and form irreversible complexes. These complexes can be purified from a complex hemolymph sample using an antibody to one of the complex members. To separate the complexes from the hemolymph, Serpin 2 antibodies were attached to protein A coated magnetic beads and then incubated with the hemolymph. Once the purified complexes and Serpin 2 were eluted, the purified proteases were identified on Orbitrap MS. In an attempt to simplify the isolation of the complexes, a magnetic bead mixing rotor column was developed to help reduce the volume of the elution to increase the concentration. This method, however, was not robust and did not improve the concentration.

Factor VIII is a protein which has therapeutic applications for the treatment of Hemophilia A. Its deficiency, either qualitative or quantitative, results in Hemophilia A, a disorder affecting approximately 1 in 10,000 males. Currently, FVIII replacement therapy uses FVIII derived from plasma or cell culture. The current cost of this therapy is in excess of $150,000 per patient per year. Thus, alternative sources that are more economical are attractive. The present work focuses upon the characterization of recombinant FVIII (rFVIII) made in the milk of transgenic pigs. Two dimensional western analysis of rFVIII obtained from pig whey showed a range of FVIII species having different isoelectric points (pI) consistent with diverse glycosylation patterns. The pI of these diverse FVIII populations were accurately predicted using theoretical calculations based upon primary protein structure as variable biantennary glycosylation patterns having 0, 1, or 2 sialic acid groups present. Kinetic limitations in the adsorption of rFVIII to anion exchange media due to the nature of the complex milk environment were observed. rFVIII was purified quantitatively using batch equilibration of whey with DEAE Sepharose. This material showed proteolytic processing that was very similar to FVIII obtained from human plasma. Based upon these results, it was postulated that a dissociation of the light (A3C1C2) and heavy (A1A2B) chain due to a lack of vWF may be responsible for the low FVIII activity. / Master of Science

Microfluidic chips have gained wide applications in various fields, including medicine, environmental sciences and forensic investigations. They are used for the separation of proteins, blood, bacterial cell suspensions, antibody solutions, and drugs. Microfluidic chips display significant advantages, which include faster analysis time, reduced amounts of samples and reagents volumes, flexibility in design and increased separation efficiency. Whole column imaging detection (WCID) exhibits significant advantages compared to other detection methods that are widely used for detecting analytes after the separation of these analytes using isoelectric focusing. With these other methods, there is a need to mobilize the focused sample bands past the detector after separation but with WCID, there is no need for mobilization step. The aim of this research is further development of WCID by characterizing microfluidic chips fabricated for the detection system, to enhance its detection so that high efficiency can be obtained. The chips were fabricated using soft lithography technology at the Microfluidic laboratory, University of Waterloo and they were used to perform isoelectric focusing of various proteins in our laboratory. The fabricated chips with straight channel design were used to carry out isoelectric focusing of some proteins and the results obtained were compared with the results obtained using commercial cartridges. The chips with tapered channel design were used to carry out isoelectric focusing of proteins in which thermally generated pH gradient principle was employed. The samples after separation were sprayed into a mass spectrometer using nano-electrospray interface to obtain their molecular masses. Compatible cartridges for nano-electrospray mass spectrometer were developed and these cartridges were used to carry out capillary isoelectric focusing of low molecular pI markers and proteins. These cartridges were also connected to the nano-electrospray mass spectrometer to obtain the mass to charge ratios of some proteins. The fabricated microfluidic chips with straight channel design were also used to investigate the interaction between drugs and protein.