Functional Proteomics and Nanotechnology-Based Microarrays

1. NANOTECHNOLOGY APPLICATIONS OF NUCLEIC ACID PROGRAMMABLE PROTEIN ARRAYS. 1.1 Introduction. 1.2 Materials and Methods. 1.2.1 The FLEXGene repository. 1.2.2 Nucleic Acid-Programmable Protein Array (NAPPA). 1.2.3 Protein-protein interactions. 1.3 Results. 1.3.1 Atomic force microscopy (AFM). 1.3.2 Nanogravimetry. 1.3.3 Mass spectrometry (MS). 1.3.4 Anodic porous alumina (APA). 1.3.5 Fluorescence via DNASER and bioinformatics. 1.3.6 Transcription translation kit. 1.4 Discussion. 1.5 Conclusions. Acknowledgments. References. 2. BIOINFORMATICS AND FLUORESCENCE DNASER FOR NAPPA STUDIES ON CELL TRANSFORMATION AND CELL CYCLE. 2.1 Introduction. 2.2 Materials and Methods. 2.2.1 The experimental layout. 2.2.2 Procedure of NAPPA expression and DNASER analysis. 2.3 Results. xvi Functional Proteomics and Nanotechnology-based Microarrays. 2.3.1 NAPPA DNASER imaging. 2.3.2 NAPPA-targeted prediction of gene interactions relevant to progressing between phases of cell cycle of human T lymphocytes. 2.3.3 NAPPA-targeted prediction of protein interactions relevant to differences between lymphoma and normal T cells. 2.3.4 Planned experimentation. 2.4 Conclusions. Acknowledgments. References. 3. LABEL FREE DETECTION OF NAPPA VIA MASS SPECTROMETRY. 3.1 Introduction. 3.2 Materials and Methods. 3.2.1 The experimental layout. 3.2.2 Fabrication of NAPPA. 3.2.3 NAPPA expression. 3.2.4 Autoflex analysis. 3.3 Results. 3.3.1 Human kinase NAPPA. 3.3.2 NAPPA for MS. 3.3.2.1 Matching algorithm. 3.4 Conclusions. Acknowledgments. References. 4. LABEL FREE NAPPA VIA NANOGRAVIMETRY. 4.1 Introduction. 4.2 Materials and Methods. 4.2.1 QCM-frequency technique. 4.2.2 QCM-D dissipation (quality) factor technique. 4.3 Results. 4.4 Conclusions and Suggestions. Acknowledgments. References. 5. LABEL-FREE NAPPA: ANODIC POROUS ALUMINA. 5.1 Introduction. 5.2 Materials and Methods. 5.2.1 Experimental details. 5.2.2 Mechanical tests for anodic porous alumina. 5.2.2.1 Grip test. 5.2.2.2 Ball-crush test. 5.2.2.3 APA over glass slides and reversibility test. 5.3 Results. 5.3.1 Electric impedance spectroscopy set up for NAPPA analysis. 5.3.2 Sponge effect and application to existing spotting systems. 5.3.3 AFM analysis of APA samples. 5.4 Conclusions. Acknowledgments. References. 6. LABEL FREE DETECTION OF NAPPA VIA ATOMIC FORCE MICROSCOPY. 6.1 Introduction. 6.2 Materials and Methods. 6.2.1 Technical details for NAPPA imaging by AFM. 6.3 Results. 6.3.1 NAPPA investigations with the functionalization of cantilever and the measurement of interaction forces. 6.4 Conclusions and Advancements. Acknowledgments. References. 7. CELL FREE EXPRESSION AND APA FOR NAPPA AND PROTEIN NANOCRYSTALLOGRAPHY. 7.1 Introduction. 7.2 APA and NAPPA Microarray. 7.3 Background of Cell-Free Protein Synthesis. 7.4 APA-Cell Free and Protein Nanocrystallography. 7.5 Materials and Methods. 7.5.1 The PURExpress system and its advantages. 7.5.2 APA template preparation. 7.5.2.1 APA process. 7.5.2.2 APA process. 7.5.3 In vitro applications of the PURExpress system. 7.5.3.1 High throughput functional genomics and Proteomics. 7.5.3.2 Protein engineering. 7.5.3.3 Study of protein expression, translation and folding. 7.5.3.4 Incorporation of unnatural amino acids. 7.5.4 Crystal growth on APA template and in nanovolume. 7.5.4.1 APA protein crystals X-ray diffraction. 7.5.4.2 Nanovolume protein crystals X-ray diffraction. 7.5.4.3 Data processing and reduction. 7.5.4.4 Nanovolume-grown protein crystal structure: LB versus classical. 7.6 Conclusions and Future Prospectives. Acknowledgments. References. 8. STRUCTURAL AND FUNCTIONAL STUDIES ON THE HELICOBACTER PYLORI PROTEOME: THE STATE OF THE ART. 8.1 Introduction. 8.2 The cag Pathogenicity Islands. 8.3 Enzymes. 8.3.1 Enzymes involved in fatty acids metabolism. 8.3.2 Enzymes of the shikimate pathway. 8.3.3 Enzymes with antioxidant properties. 8.3.4 Enzymes involved in sugar metabolism. 8.3.5 Enzymes involved in cell wall biosynthesis. 8.3.6 Vitamins precursors. 8.3.7 Enzymes involved in NAD biosynthesis. 8.3.8 Enzymes involved in peptides biosynthesis or degradation. 8.3.9 Urea metabolism. 8.3.10 Miscellanea. 8.4 Proteins Interacting with DNA and Regulators of Gene Expression. 8.5 Toxins and Other Proteins Directly Involved in Pathogenicity. 8.6 Unknown Function Proteins. 8.7 Conclusions. References. 9. OVERALL PROTEOME ALTERATIONS DURING REVERSE TRANSFORMATION OF GROWING CHO-K1 CELLS. 9.1 Introduction. 9.2 General Effects of cAMP on CHO-K1 Cells. 9.2.1 Involvement of vimentin. 9.2.2 cAMP-induced gene expression. 9.2.3 Puck model. 9.3 Overall CHO Proteome Investigation. 9.3.1 LC-ESI MS analysis of nucleus fraction. 9.3.2 HPLC analysis. 9.3.3 Gel electrophoresis. 9.3.4 MALDI-TOF MS. 9.4 Conclusions. References. 10. ORGAN TRANSPLANTS AND GENE MICROARRAYS. 10.1 Introduction. 10.2 A Glass of Genes? 10.2.1 Microarray principle. 10.2.2 Sample source. 10.2.3 Overview of microarray analysis in organ transplantation. 10.3 Organ Transplantation Under Gene Magnifying Glass. 10.3.1 Pre- or peri-transplantation related factors. 10.3.2 Microarray analysis in acute allograft rejection. 10.3.3 Microarray analysis in chronic allograft rejection. 10.3.4 Immunosuppressive drugs in transplantation. 10.4 Conclusion. References. Abbreviations. 11. SIGNALING NETWORKS. SIMULATIONS OF BIOCHEMICAL INTERACTIONS. APPLICATIONS TO MOLECULAR ONCOLOGY. 11.1 Introduction. 11.2 Molecular Interaction Maps (MIMs). 11.3 Implementation of a Dynamic Modeling of Cell Signaling Pathways and Networks: What It Is About? What Do We Expect From Dynamic Modeling? 11.3.1 Model construction is error prone. 11.3.2 Stochastic simulation approach. 11.3.3 Sufficient/adequate MIM size. 11.3.4 Forward and backward kinetics. 11.3.5 Problem of validation of a given dynamic simulation. 11.3.6 Multidimensional sensitivity analysis. 11.4 Conclusions. References. 12. LABEL-FREE DETECTION OF NAPPA: SURFACE PLASMON RESONANCE. 12.1 Nucleic Acids Programmable Protein Arrays. 12.2 Surface Plasmon Resonance. 12.2.1 What is a Surface Plasmon and what is Surface Plasmon resonance? 12.2.2 The principle of measurement-towards a sensorgram. 12.3 Materials and Methods. 12.3.1 NAPPA array production. 12.3.2 NAPPA expression. 12.3.3 Detection of in situ expressed protein by SPR. 12.4 Results. 12.4.1 Detection of GST purified protein by SPR. 12.4.2 Detection of in situ expressed protein by SPR. 12.5 Conclusions and Future Directions. References. Index.