Thesis (M.S.)--Central Connecticut State University, 1999. / "In partial fulfillment of the requirements for the degree of Master of Science [in Art.]."

På beställning av GE Healthcare AB har en sammanställning gjorts över interaktionen mellan metalljoner och kelatorer samt metalljoner och proteiner vid immobiliserad metalljonsaffinitetskromatografi (IMAC). Kelatorer är kemiska föreningar som kan användas för att binda metalljoner. Denna sammanställning kan användas som en vägledning för optimering av IMAC. Det är ett vedertaget faktum att bindningsstyrkan mellan metalljoner och proteiner, bland annat, beror på hur många bindningsställen på metalljonen som finns kvar för proteiner att binda till. Desto fler bindningsställen kelatorn upptar på metalljonen, desto svagare blir interaktionen med proteinet. Denna sammanställning är begränsad till ett antal metalljoner, som till exempel Cu2+, Ni2+, Co2+, Zn2+ och Fe3+, och kelatorerna ”Iminodiacetic acid” (IDA), ”tris(2-aminoethyl)amine” (TREN), ”1,4,7-triazacyclononane” (tacn), ”tris(carboxymethyl)ethylenediamine” (TED) och ”nitrilotriacetic acid” (NTA). Några intressanta skillnader i bindningsstyrkan mellan ett antal intermediära metalljoner, kopplade till en kelator, och proteiner har framkommit i denna studie. Dessa relationer, för kelatorerna IDA, TREN och tacn, finns redovisade nedan (starkast bindningsaffinitet längst till vänster). IDA: Cu2+ > Ni2+>Zn2+>Co2+ TREN: Cu2+ > Ni2+ Tacn: Cu2+ > Ni2+ > Zn2+ > Co2+ > Cr2+ ≈ Mn2+

Documento de trabajo que permite reconocer y aplicar las funciones básicas en el uso del iMac en los diferentes campus de la Universidad Peruana de Ciencias Aplciadas (UPC).

In this work, the synthesis and characterization of functionalized biosurfaces that can be used for bioseparations and bio-nanotechnology are reported. A novel protein purification technique that incorporates chelating ligands and polymers onto the same chromatographic matrix is explored. A polysaccharide based gel, agarose, was modified systematically with hybrid ligands of the chelator iminodiacetic acid (IDA) and the polymer polyethylene glycol (PEG). The PEG molecule acts as a blocking polymer that can allow only small proteins to permeate onto the matrix surfaces and form conventional immobilized metal ion affinity chromatographic (IMAC) interactions with the chelators. Kinetic studies of chelator and polymer attachment were performed in order to effectively control the chelator and polymer densities on the matrix. Studies with different PEG surface densities and their effects on the adsorption of several proteins (e.g. myoglobin, lysozyme and bovine serum albumin (BSA)) were evaluated to characterize these new hybrid size exclusion IMAC (SEIMAC) matrices. An exclusion effect was observed while adsorption as observed in IMAC systems took place.Functionalization schemes and procedures were extended in the activation and incorporation of affinity ligands on inorganic surfaces such as gold surfaces. Functional gold platforms were explored for development of nano-interconnects via functionalized self assembled monolayers (FSAMs) on gold to attach specific affinity ligands as linkers to immobilize biomolecules, such as microtubules (MTs). MTs eventually could be utilized as self assembling structures and templates for fabrication of nano-scale bio-interconnect arrays and networks. In this work, different organothiols were used to form FSAMs and anti-glutathione S-transferase was attached as a linker to utilize the attachment of MT cap proteins, gamma-tubulin. The gamma-tubulin could recognize specifically a heterodimer of the MTs and can provide a nucleation center for MT growth. Several methodologies were employed including photolithographic methods and the use of photoreactive compounds for proper micro/nano scale dual protein functionalization of surfaces with homogeneous affinity ligands and with heterogeneous ligands as well.

No description available.

The method for the study of protein phoshorylation sites was elaborated. This method is based on the IMAC separation of phosphopeptides from protein proteolytic digests using new magnetic sorbents and on their subsequent identification by mass spectrometry (MS). Magnetic non-porous hydrophilic poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) particles prepared by the dispersion polymerization and modified with iminodiacetic acid (IDA) with immobilized Fe(III) and Ga(III) ions were employed for the enrichment of phosphopeptides from the proteolytic digests of two model proteins, porcine pepsin A and bovine α-casein. The optimum conditions for phosphopeptide adsorption and desorption in both cases were investigated and compared. The phosphopeptides separated from both proteolytic digests were analyzed by matrix-assisted laser desorption/ionization time-of-flight MS. For the immunochemical separation of phosphoproteins, protein fraction containing antibodies was obtained from egg yolk of hens immunized with O-phosphoryl-L-serine conjugated to key limpet hemocyanin. Antibodies were purified using affinity chromatography on immobilized α-casein and their presence was proven by MS. Specificity of the obtained antibodies was examined using ELISA tests. Obtained results showed, that specificity...

Les laboratoires sur puce sont des dispositifs miniaturisés qui offrent la possibilité d'intégrer en ligne toutes les étapes de la chaîne analytique tout en réduisant les volumes d’échantillon et les temps d’analyse. Ainsi, ils constituent potentiellement un outil de diagnostic particulièrement adapté pour l’analyse de biomarqueurs phosphorylés, pour lesquels une préconcentration est nécessaire en raison de leur faible abondance dans les fluides biologiques. C’est pourquoi, de nouvelles méthodes, dédiées à l'enrichissement de phosphopeptides, ont été développées ces dernières années et en particulier celles utilisant des supports solides basées sur la chromatographie d’affinité sur des ions métalliques immobilisés (IMAC). Parmi les supports solides intégrables en microsystème, les monolithes organiques constituent une option privilégiée grâce à la possibilité d’être synthétisés in situ. Le but de ce travail de thèse était donc de développer un laboratoire sur puce intégrant une préconcentration des phosphopeptides sur support monolithique basé sur le principe de l’IMAC et leur séparation électrophorétique en ligne.Dans un premier temps, nous avons développé deux approches innovantes qui ont permis de synthétiser pour la première fois un monolithe à base d’éthylène glycol méthacrylate phosphate (EGMP) et de bisacrylamide (BAA) par voie photochimique dans des microsystèmes. La première stratégie développée dans des puces en verre repose sur la synthèse du monolithe à l’aide d’un microscope à épifluorescence. La deuxième approche est basée sur les propriétés photochimiques d’un nouvel amorceur qui a permis de synthétiser et d’ancrer le monolithe, en une seule étape, aux parois des puces en polydiméthylsiloxane (PDMS). Une caractérisation de ce monolithe en termes de morphologie, de perméabilité, de porosité et de surface spécifique a ensuite été réalisée. Ceci a permis de démontrer le potentiel de ce monolithe pour la préconcentration.Dans un deuxième temps, une méthode de séparation par électrophorèse couplée à une détection par fluorescence a été développée sur puce en verre. Celle-ci a permis de séparer un mélange de phosphopeptides modèles fluorescents possédant différents sites et degrés de phosphorylation. Les phosphopeptides ont été détectés en moins de 2 min avec une excellente résolution (R>3) et une bonne efficacité (plateaux théoriques compris entre 11000 et 25000). Enfin, le couplage en ligne du module de préconcentration monolithique et de séparation/détection a été réalisé. Sur ce dispositif miniaturisé, une préconcentration basée sur l’IMAC-Zr4+ a ainsi été développée. L’efficacité de la capture et de l’élution des phosphopeptides a été démontrée et des facteurs de préconcentration supérieurs à 340 ont été obtenus. En conclusion, ce laboratoire sur puce ouvre des perspectives très prometteuses dans le domaine du diagnostic de pathologies dont le processus physiopathologique implique des phosphopeptides.Mots clés : laboratoire sur puce, microsystème, phosphopeptide, IMAC, monolithe, photopolymérisation, préconcentration, électrophorèse sur puce / A lab on-a-chip is a miniaturized device that integrates onto a single chip different analytical steps (preconcentration, separation, detection...) with minimal sample consumption and short analysis time. They are potentially beneficial in phosphorylated biomarker analysis for which a preconcentration step is necessary because of their low abundance in biological fluids. That's why selective enrichment methods of phosphopeptides have been developed in recent years in particular those based on solid supports like Immobilized Metal Affinity Chromatography (IMAC). Among the solid supports, organic polymer monoliths present practical advantages when used in microchips due to their ease of preparation and in situ polymerization. The aim of this work was to develop a lab-on-a-chip integrating a monolithic support for online IMAC-based preconcentration and electrophoretic separation of phosphopeptides.In the first part, we developed two innovative approaches which allowed us to synthesize, for the first time, an ethylene glycol methacrylate phosphate-co-bisacrylamide (poly (EGMP-co-BAA)) monolith by a photo-driven process in microsystems. The first monolith synthesis approach was developed in glass microchannels using an inverted epifluorescence microscope as UV-irradiation source. The second approach was based on the photochemical properties of a new initiator which allowed the simultaneous synthesis and anchorage of the monolith in native polydimethylsiloxane (PDMS) microchips. A characterization (morphology, permeability, porosity and specific surface area) of (poly (EGMP-co-BAA)) monolith was then performed which demonstrated the potential of this monolith for preconcentration.Then a glass microchip electrophoresis method coupled to a detection by fluorescence was developed to separate a mixture of phosphopeptides fluorescent models differing with the position and number of phosphorylation sites. The phosphopeptides were detected in less than 2 min with excellent resolution (R> 3) and good efficiencies ranging from 11000 to 25000 plates. Finally, an integrated microdevice was developed by combining online preconcentration based on IMAC-Zr4+ and separation/detection of phosphopeptides. The performance of this integrated microdevice to capture and to elute the phosphopeptides was demonstrated and signal enhancement factors (SEF) higher than 340 were obtained. This lab-on-a-chip device opens news perspectives for phosphoproteomic applications and the diagnostic of diseases where the pathophysiological process involves phosphopeptides

The main objective of this research was to develop a protocol in which pyridoxal phosphate (PLP) would act as a tag to identify PLP-dependent enzymes from complex mixtures or cell lysates. Following the purification of a PLP-dependent enzyme (CysM), a method was developed to reduce the PLP-lysine Schiff base to form a chemically stable bond between the PLP and the protein. The reduced protein was enzymatically digested resulting in multiple peptide fragments with one or more containing PLP (bound to the active site lysine). These fragments were analyzed by monitoring the absorbance or fluorescence using High Performance Liquid Chromatography. Immobilized Metal Ion Affinity Chromatography (IMAC) was then used to enrich the PLP-peptide(s) from the peptide mixture. The PLP-bound peptide(s) was then analyzed using Liquid Chromatography-Mass Spectrometry (LC-MS). More specifically, sodium borohydride (NaBH4) was used to reduce the Lysine-PLP bond in CysM. This reaction was monitored by either UV-vis spectroscopy or mass spectrometry. Trypsin was used to enzymatically digest the reduced CysM before it was enriched with IMAC and analyzed with LC-MS. Since the objective of this project was to develop a method which could be applied to a cell lysate, IMAC was used as an enrichment method to separate the PLP-peptide(s) from other peptides within the mixture. The PLP-peptide(s) was then located in the peptide mixture by monitoring the absorbance at 325 nm. The LC-MS results of the full reaction before IMAC treatment versus the final column, when monitoring the mass spectrum, showed that the treatment using the IMAC column separated the PLP-peptides from all other peptides within the sample. Using IMAC to enrich specifically the PLP-peptides, followed by analysis with LC-MS, may be a useful method for studying PLP-dependent enzymes within the proteome or the "PLP-ome."

Ebolavirus (EBOV) is a single stranded RNA virus that causes haemorrhagic fever in humans and other mammals. The EBOV encodes 7 proteins, NP, L, VP30, VP35, VP40, GP and VP24. VP24 is believed to be one of the EBOV proteins that causes the extreme virulence of the pathogen. The protein blocks the interaction between PY-STAT1 and KPNA, a protein that is involved in the import of PY-STAT1 into the nucleus. The nuclear import of PYSTAT1 is therefore blocked. This leads to the inhibition of IFN signalling. The purpose of this study was to express and purify VP24 and KPNA5. The proteins recombinantly expressed as a fusion tag in E. coli in lysogeny broth. Purification of VP24 was done using immobilized metal ion affinity chromatography, size exclusion chromatography and ion exchange chromatography. Characterization of the protein was analysed using circular dichroism. The results obtained from this study showed that VP24 could be purified in pH 10 buffers with little loss of protein due to aggregation and the protein was folded with an alphahelical structure. The expression and purification of KPNA5 was more complicated and further evaluation is left for future studies. The established protocol for expression and purification of VP24 and the initial work on KPNA5 will go a long way to aiding future studies on the system, thus the answer to the question regarding the extreme virulence of EBOV will be closer.

La phosphorylation de protéines est un régulateur clé de voies de signalisation cellulaire. Elle est impliquée dans la plupart des événements cellulaires et contrôle les processus biologiques tels que la prolifération, la différenciation et l'expression des gènes. Une phosphorylation anormale de protéines peut être observée dans diverses maladies comme certains cancers ou maladies neurodégénératives. Ces protéines constituent donc des biomarqueurs potentiels pour le développement d’outils de diagnostic. Cependant les phosphoprotéines peuvent être présentes à faibles concentrations dans les liquides biologiques et des techniques d’enrichissement sélectif des protéines phosphorylées doivent être développées en amont des analyses. L'une des approches les plus courantes est basée sur la chromatographie d'affinité de type IMAC. Le but de ce travail de thèse était de développer un microsystème contenant un monolithe en tant que support solide d'extraction pour réaliser une préconcentration sélective de phosphopeptides par IMAC. La polymérisation par UV et la caractérisation (perméabilité, porosité et surface spécifique) d'un monolithe à base de phosphate de méthacrylate d'éthylène glycol dans des capillaires de silice ont été d'abord réalisées. Puis, nous avons tenté d'optimiser les différentes étapes de l’IMAC (immobilisation du métal, chargement de l’échantillon, lavage et élution). Une immobilisation efficace de zirconium sur le monolithe phosphaté a été démontrée par des mesures de FEO dans un capillaire et a été par la suite confirmée par la rétention d'un phosphopeptide modèle. Nous avons démontré que le monolithe phosphaté était également un support d’échange de cations vis-à-vis de peptides fortement basiques. Les protocoles de chargement et d'élution ont également été étudiés, mais nécessitent encore d’être améliorés. La transposition de l'enrichissement de phosphopeptides par IMAC sur un système miniaturisé a ensuite été envisagée. Nous avons choisi deux matériaux pour la puce : le PDMS, qui est un polymère attractif pour son faible coût, sa facilité de microfabrication, ses excellentes propriétés en termes de biocompatibilité ainsi que ses nombreuses possibilités d'intégration (enrichissement, séparation, détection) et le verre plus communément employé pour développer des microsystèmes analytiques et possédant une bonne transparence aux UV. Toutefois, le PDMS présente deux inconvénients majeurs: son absorption élevée et sa perméabilité importante à l'oxygène qui inhibe la polymérisation radicalaire. A l’exception de quelques tentatives, ce matériau n'a jamais été employé avec succès comme support pour la polymérisation d’un monolithe. Afin de pouvoir surmonter ces problèmes, nous avons étudié plusieurs stratégies de traitement de surface du PDMS tels que le traitement par plasma d’oxygène ou encore le revêtement au borosilicate. Enfin, nous avons démontré que notre module d’IMAC fonctionnait correctement dans un microsystème en verre. Ce module miniaturisé devrait à l’avenir s’intégrer dans un microsystème d’analyse dédié au diagnostic de la maladie d'Alzheimer. / Protein phosphorylation is a key regulator of cellular signaling pathways. It is involved in most cellular events and strictly controls biological processes such as proliferation, differentiation and gene expression. An abnormal phosphorylation can be observed in various diseases such as some cancers or neurodegenerative diseases. Therefore, these proteins are potential biomarkers for the development of diagnostic tools. However, phosphoproteins can be present at low abundance in biological samples and selective enrichment techniques have to be developed prior to the analysis process. One of the most common approaches is based on Immobilized metal affinity chromatography (IMAC). The goal of this work was to develop a microsystem which contains a porous polymer monolith (PPM) as a solid phase extraction for a selective preconcentration of phosphopeptides by IMAC. UV-polymerization and characterization (permeability, porosity and specific area) of a monolith based on ethylene glycol methacrylate phosphate in silica capillaries was first performed. Then, we tried to optimize the different IMAC steps (metal immobilization, sample loading, washing and elution). An efficient immobilization of zirconium on the phosphated PPM was demonstrated by EOF measurements in capillary and confirmed by retention of a model phosphopetide. We demonstrated that the phosphated monolith was also a strong cation exchanger of highly basic peptides. Protocols of loading and elution were also studied but need to be further optimized. Transposition of phosphopeptides enrichment by IMAC on a miniaturized system was then considered. We selected two microchip materials: PDMS is an attractive polymer for its low cost, its ease of microfabrication, its excellent working properties (biocompatibility, UV transparent with low autofluorescence) and many integration possibilities (enrichment, separation and detection) and glass microchip more common and having a good UV transparency. However, PDMS presents two major disadvantages: high absorption property, and oxygen permeability which quench free radical polymerization. Except a few attempts, this material has not been employed successfully as mould for monolith polymerization. To overcome these problems, we investigated several strategies for PDMS surface treatments such as plasma treatment and borosilicate coating. Finally, we demonstrated that our IMAC module performed well on glass microchip. This miniaturized module should be integrated in the future into a microsystem dedicated to the diagnosis of Alzheimer disease.