A high throughput proteomic analysis method is described here that uses more economically favorable, easily manufactured probe surface that can be directly incorporated on the MALDI target. On-Probe Affinity Capture (OPAC) MALDI is a method that uses the RF pulsed plasma modified target surfaces for the protein purification, separation and identification all on the same single probe and one of the highest advantage of this method is the number of different experiments that can be carried out simultaneously using the intelligent design of the probe. The new design of the OPAC probe presented in this dissertation gives the ability to perform about 100 different experiments on one single MALDI target. These OPAC probes can be used for the fractionation and analysis of proteins from complex biologically derived samples. The separated proteins can be identified on the OPAC probe using it directly as the MALDI target and selecting a proper elution solution that depends on the chemistry of the OPAC probe, the surface bound proteins can be eluted and incorporated into the matrix crystal. This dissertation focuses mainly on developing this method for analysis of different samples. A tryptic digest of a single protein was separated and identified by submitting the peak lists to MASCOT database search and the sequence coverage obtained before and after fractionation has been compared. Then a mixture of tryptic peptides of five different known proteins were fractionated on OPAC surfaces and the identification of proteins obtained was compared before and after fractionation. Further developing this technique, biologically derived mixtures of proteins from two different well studied sources have been analyzed using OPAC-MALDI. Escherichia coli bacterial proteome was digested and fractionated and the peptides were studied using De Novo sequencing method and their affinity fractionation behavior is confirmed by calculating the iso electric point (pI) and the hydrophobicity of the predicted peptide sequence. Synechosystis sp PCC 6803 was cultured and the protein extracts were prepared for the OPAC studies. The clear fractionated of this mixture was observed and the amount of information derived after fractionation is found to be significantly higher than the unfractionated sample. Taking a slightly different approach, a phosphoprotein binding OPAC probe was prepared using commercially available poly(methyl methacrylate) (PMMA) film. The hydrolyzed PPMA films were reacted with CuCl2 solution to incorporate metal ions on the surface by electrostatic interaction, which then facilitates the phosphoprotein binding on the OPAC probe. This was demonstrated using a binary mixture of commercially available peptides and fractionating the mixture on Cu-impregnated PMMA film. Finally, in a collaborative work, the possibility of increased surface binding capacity was explored by using a synthetic organic nanosponge surface that expands and collapses due to change of pH. These brush polymers were prepared by Dyer group and a binary mixture of peptides were fractionated and analyzed by MALDI MS.

No description available.

High performance liquid chromatography (HPLC) is one of the major techniques in separat-ion sciences. Faster separation and higher efficiency are required to meet ever-growing demands. Despite numerous studies and achievements on improving mass transfer in particulate packings discontinuity seems to be the cornerstone drawback in their development. Macroporous continuous beds or monoliths are therefore a promising alternative to the particle medium. This thesis deals with preparation of new monoliths used as carrier for HPLC. Two different approaches were developed for two polymer systems. One was based on polycondensation of epoxy resins and polyamines which were components of an oil-in-water emulsion. An epoxy resin mixture was dispersed in aqueous polyamine phase with the aid of a surfactant. The other involved a traverse of a ready-made polymer solution around its upper critical solution temperature (UCST). In other words, linear polyamides, non-covalently crosslinked polymers, dissolved in a solvent at temperature higher than their UCST followed by slow cooling to below the critical temperature to precipitate the polymers. Partly re-established hydrogen bonds resulted in the formation of crystallites that interconnected into a network structure. Factors controlling morphology and porosity of final products were investigated. The study also deals with surface modifying for chromatographic applications. Functionalization pathways attempted in the thesis were quaterization of inherent amine of the epoxy-based monoliths and grafting tentacle ion groups via glycidyl methacrylate by atom transfer radical polymerization (ATRP) for ion exchange chromatography (IEC).

The aim of presented diploma thesis was to characterize recent knowledge in the field of beer proteomics. The main part of this work was focused on modern instrumental methods of protein analysis, especially on protein identification by mass spectrometry. In experimental part proteins from selected beer samples were isolated, purified and separated by 2-D electrophoresis. The identification was performed by MALDI MS/MS and LC-MS/MS. Identified proteins were divided into 6 groups - serpines and protein Z, trypsine/-amylase inhibitors, yeast proteins, LTP protein, hordeins and other proteins. Proteomic analysis provided identification of proteins important for final analytical and sensory characteristics of the beer - for final beer quality and taste

Isoelectric trapping (IET) has become an accepted preparative-scale electrophoretic separation technique. However, there are still a number of shortcomings that limit its utility. The performance of the current preparative-scale IET systems is limited by the serial arrangement of the separation compartments, the difficulties in the selection of the appropriate buffering membranes, the effect of Joule heating that may alter separation selectivity and a lack of methods for the determination of the true, operational pH value inside the buffering membranes. In order to bolster the current membrane pH determination methods which rely on the separation of complex ampholytic mixtures, a fluorescent carrier ampholyte mixture was synthesized. The use of a fluorescent mixture allows for a reduced load of carrier ampholytes, thereby reducing a possible source of error in the pH determinations. A mixture of carrier ampholytes tagged with an alkoxypyrenetrisulfonate fluorophore was shown to have suitable fluorescence and ampholytic properties and used to accurately determine the pH of high pH buffering membranes under actual IET conditions. In a more elegant solution to the difficulties associated with pH determinations, a method utilizing commercial UV-transparent carrier ampholytes as the ampholyte mixture to be separated was developed. By using commercial carrier ampholytes and eliminating the need to synthesize, purify, and blend fluorescently tagged ampholytes, the new method greatly simplified the determination of the operational pH value of the buffering membranes. In order to address the remaining limitations, a new system has been developed that relies on (i) parallel arrangement of the electrodes and the collection compartments, (ii) a directionally-controlled convection system for the delivery of analytes, (iii) short anode-to-cathode distances, (iv) short intermembrane distances, and (v) an external cooling system. This system has been tested in four operational modes and used for the separation of small molecule ampholytic mixtures, for the separation of protein isoforms, and direct purification of a target pI marker from a crude reaction mixture.

<p>Liquid chromatography coupling with mass spectrometry (LC/MS) plays an important role in pharmaceutical characterization because of its ability to separate, identify, and quantify individual compounds from the mixture. Polymer brush layer bonded to the silica surface is designed as a novel stationary phase to improve the LC resolution and MS compatibility. The polymer thickness can be controlled to shield the analyte from interacting with the active silanol on the surface and reduce peak tailing. The functional group of the polymer can be changed to tune the selectivity in different separation modes. </p><p> </p><p>Two projects on LC/MS method development for biomolecule characterization using polymer-shell column are discussed in this work. In the first project, a polymer-shell column is used for disulfide bonds and free thiol subspecies identification, which is a major type of structural heterogeneities in IgG1. Compared with commercial columns, the polymer-shell column is able to resolve the free thiol variants without the presence of trifluoroacetic acid and greatly improve the MS signal. In the second project, a polymer-shell column is used for characterizing the drug-loading profile for antibody-drug-conjugates (ADC) via online LC/MS. The separation employs a mobile phase of 50 mM ammonium acetate to keep the ADC intact, and a gradient of water/isopropanol for ADC elution. MS data show that all ADC species remained intact and native on the column. Positional isomers can be separated and identified with the new method as well. Furthermore, to understand the surface chemistry and protein separation behavior quantitatively, a chromatographic simulation study is performed. The result shows that protein separation in RPLC can be described by a bi-Langmuir adsorption isotherm with mixed-mode retention of strong and weak sites. Smaller fractions and lower equilibrium constant of the strong site, which is the active silanol, give less tailing for protein separation.</p>