Note: Microcharacterization of Proteins -- Contents -- Section I: Overview -- I.1 Microcharacterization of Proteins -- 1 General Aspects -- 2 From a Cell to a Protein Sequence -- 3 Genome and Proteome -- Section II: Microseparation Techniques and Sample Preparation -- II.1 Microseparation Techniques I: High Resolution Gel-Electrophoretic Techniques : Qualitative, Quantitative and Micropreparative Applications -- 1 Introduction -- 2 Theory -- 3 Media and Equipment -- 3.1 Media -- 3.2 Equipment -- 4 Gel Electrophoretic Methods -- 4.1 Disc Electrophoresis -- 4.2 Gradient Gel Electrophoresis -- 4.3 Additives in Electrophoresis -- 4.4 SDS Electrophoresis -- 4.5 Isoelectric Focusing -- 4.6 Two-Dimensional Electrophoresis (2 -DE) -- 5 References -- II.2 Microseparation Techniques II: Gel Electrophoresis for Sample Preparation in Protein Chemistry -- 1 Introduction -- 2 Denaturing Techniques -- 2.1 Commonly Used SDS-Polyacrylamide Gel Electrophoresis Techniques for Protein Separation -- 2.2 Blue-SDS-PAGE for Recovery of Membrane Proteins from Gels -- 2.3 Electroelution of Proteins after Blue-SDS-PAGE -- 2.4 Electroblotting of Blue and Colorless SDS Gels -- 2.5 Isoelectric Focusing in the Presence of Urea -- 3 Native Techniques -- 3.1 Colorless-Native-PAGE -- 3.2 Blue-Native-PAGE -- 3.3 Native Isoelectric Focusing -- 4 References -- II.3 Microseparation Techniques III: Electroblotting -- 1 Introduction -- 2 Electroblotting -- 2.1 Polyacrylamide Gel Electrophoresis -- 2.2 Blot Systems -- 2.2.1 Tank Blotting -- 2.2.2 Semidiy Blotting -- 2.3 Blotting Parameters -- 2.3.1 The Blotting Process -- 2.3.2 Transfer Buffers -- 2.3.3 Addition of SDS -- 2.3.4 Addition of Methanol -- 2.3.5 Influence of Protein Concentration -- 3 Blotting Membranes -- 4 References -- II.4 Microseparation Techniques IV: Analysis of Peptides and Proteins by Capillary Electrophoresis. , 1 Introduction -- 2 Theory -- 2.1 Capillary Isotachophoresis -- 2.2 Capillary Zone Electrophoresis -- 2.3 Electroosmotic Flow -- 3 Instrumentation -- 3.1 Injection -- 3.2 Detection -- 4 Applications -- 4.1 Peptide Separations -- 4.2 Protein Separations -- 5 References -- II.5 Microseparation Techniques V: High Performance Liquid Chromatography -- 1 Introduction -- 2 Principle of HPLC -- 3 Getting Started -- 3.1 Solvents -- 3.2 Pump -- 3.3 Gradient -- 3.4 Pre-Column Split -- 3.5 Sample Preparation -- 3.6 Injector -- 3.7 Tubings -- 3.8 In-Line Filter, Guard Column -- 3.9 Column -- 3.10 Detection -- 3.11 Fractionation -- 4 Applications -- 5 References -- II.6 Sample Preparation I: Removal of Salts and Detergents -- 1 Introduction -- 2 Removal of Salts or Polar Components -- 2.1 Protein-Binding Membranes -- 2.2 High Performance Liquid Chromatography -- 2.3 Desalting on Microcolumns -- 3 Removal of Detergents and Apolar Contaminants -- 3.1 Detergents -- 3.2 Protein-binding Membranes -- 3.3 Precipitation -- 3.4 SDS-PAGE -- 3.5 Concentrating Gels -- 4 "Golden Rules" for Protein and Peptide Handling -- 5 References -- II.7 Sample Preparation II: Chemical and Enzymatic Fragmentation of Proteins -- 1 Strategy -- 2 Denaturation, Reduction and Alkylation -- 3 Enzymatic Fragmentation -- 3.1 Enzymes -- 3.2 Practical Considerations -- 3.2.1 Practical Considerations for On-Membrane Digestions -- 3.2.2 Practical Considerations for In-Gel Digestions -- 3.2.3 Automation of Digest Procedures -- 4 Chemical Fragmentation -- 4.1 Cyanogen Bromide Cleavage -- 4.2 Partial Acid Hydrolysis -- 4.3 Hydroxylamine Cleavage of Asn-Gly Bonds -- 4.4 Cleavage at Tryptophan -- 4.5 Cleavage at Cysteine -- 5 References -- Section III: Bioanalytical Characterization -- III.1 Amino Acid Analysis -- 1 Introduction -- 2 Sample Preparation -- 2.1 Peptides and Proteins. , 2.1.1 Enzymatic Hydrolysis -- 2.1.2 Acid Hydrolysis -- 2.1.3 Alkaline Hydrolysis -- 2.2 Free Amino Acids -- 3 Derivatization -- 3.1 Post-Column Derivatization -- 3.1.1 Ninhydrin -- 3.1.2 Orthophthaldialdehyde -- 3.1.3 Fluorescamine -- 3.2 Pre-Column Derivatization -- 3.2.1 Phenylisothiocyanate -- 3.2.2 Orthophthaldialdehyde -- 3.2.3 Fluorenylmethyl Chloroformate -- 3.2.4 Dabsyl Chloride -- 3.2.5 Dansyl Chloride -- 3.2.6 Chiral Reagents -- 4 Data Evaluation -- 5 Instrumentation -- 6 Discussion -- 7 References -- III.2 Chemical Methods for Protein Sequence Analysis -- 1 The Edman Degradation -- 1.1 Coupling, Cleavage and Conversion -- 1.2 Identification of the PTH Amino Acids -- 2 Instrumentation -- 2.1 The Liquid Phase Sequencer -- 2.2 The Solid Phase Sequencer -- 2.3 The Gas Phase Sequencer -- 2.4 The Pulsed Liquid Phase Sequencer -- 2.5 The Biphasic Column Sequencer -- 3 Difficulties of Amino Acid Sequence Analysis -- 3.1 The Sample and Sample Matrices -- 3.2 Difficulties with the Edman Chemistry -- 4 C-Terminal Sequence Analysis -- 4.1 Chemical Degradation -- 4.2 Enzymatic Methods -- 4.3 Methods Combined with Mass Spectrometry -- 5 References -- III.3 Analyzing Post-Translational Protein Modifications -- 1 Introduction -- 2 Classification of Post-Translational Modifications According to their Behavior during Purification and Edman Degradation -- 2.1 Modifications: Stable during Purification and Edman Degradation -- 2.2 Modifications : Stable during Purification but Unstable during Edman Degradation -- 2.3 Modifications : Unstable during Purification and Edman Degradation -- 3 Examples -- 3.1 1-Methyl-Histidine -- 3.2 Glyco-Asparagine, Glyco-Threonine -- 3.3 Phospho-Tyrosine -- 3.4 N-Pyruvyl or N-a-Oxo-Butyric Acid -- 3.5 Gluco-Arginine -- 3.6 Farnesyl-Cysteine -- 3.7 Phospho-Serine -- 3.8 Phospho-Threonine. , 3.9 Screening for Phospho-Serine/Threonine Containing Peptides by HPLC/MS -- 3.10 Lanthionine, 3 -Methyl-Lanthionine, Dehydroalanine, Dehydro-a-aminobutyric Acid -- 4 References -- III.4 Analysis of Biopolymers by Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry * -- 1 Introduction -- 2 Development of MALDI -- 2.1 Mechanism of Matrix-Assisted Laser Desorption/Ionization -- 3 Instrumentation -- 3.1 Time-of-Flight (TOF) Mass Spectrometers -- 3.2 Laser Desorption Ion Source -- 3.3 Ion Detection and Data Collection -- 4 Applications -- 4.1 Sample Preparation -- 4.2 Ion Fragmentation -- 4.3 Molecular Weight Determination of Proteins and Glycoproteins -- 4.3.1 Accuracy of Mass Determination -- 4.3.2 Sensitivity and Mass Range -- 4.4 Analysis of Oligonucleotides -- 4.5 Analysis of Glycans and Glycoconjugates -- 5 Combination of MALDI with Biochemical Methods -- 5.1 Peptide Mapping of Digested Proteins by MALDI -- 5.2 Combination of MALDI and Gel Electrophoresis -- 5.3 Combination of MALDI with Capillary Zone Electrophoresis -- 6 References -- III.5 MALDI Postsource Decay Mass Analysis -- 1 Introduction -- 2 Methodology and Principal Mechanisms of Postsource Decay -- 2.1 Internal Energy Uptake and Ion Stability -- 2.2 Instrumentation for MALDI-PSD Analysis -- 2.3 Precursor Ion Selection -- 3 Applications and Spectra Interpretation -- 3.1 Sequence Analysis of Peptides -- 3.2 Primary Structure Analysis of Unknown Peptides -- 5 References -- 3.3 Primary Structure Analysis of Modified Peptides and Other Biomolecules -- 4 Potential and Perspectives -- III.6 Electrospray Mass Spectrometry -- 1 Introduction -- 2 Instrumentation -- 2.1 The Electrospray Source -- 2.2 The Mass Analyzer -- 2.3 The Detector -- 3 Mass Spectra of Proteins -- 4 Coupling of Chromatographic Methods to the Mass Spectrometer -- 4.1 On-Line HPLC-MS. , 4.2 Coupling of a Protein Sequencer to an ESI Mass Spectrometer -- 4.3 Microcapillary LC Coupled to Mass Spectrometry -- 4.4 Capillary Electrophoresis Coupled to Mass Spectrometry -- 5 Purity Control of Synthetic Peptides -- 5.1 Sample Introduction with an Autosampler -- 5.2 Off-Line HPLC-MS -- 5.3 Characterization of Combinatorial Compound Collections -- 6 References -- III.7 Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR-MS) -- 1 Introduction -- 2 Principle -- 2.1 Resolution, Mass Accuracy, Mass Range and Sensitivity -- 2.2 Tandem Mass Spectrometry -- 3 Electrospray and FT-ICR-MS -- 4 References -- III.8 Sequence Analysis of Proteins and Peptides by Mass Spectrometry -- 1 Introduction -- 2 Basics in Peptide Fragmentation -- 3 Instrumentation and Generation of MS/MS Data Sets -- 3.1 MALDI-PSD Time-of-Flight Mass Spectrometry -- 3.2 ESI-Triple Stage Quadrupole Mass Spectrometry -- 3.3 ESI-Ion Trap Mass Spectrometry -- 3.4 ESI-Quadrupole-TOF Mass Spectrometry -- 4 Coupling Methods for Mass Spectrometry -- 4.1 HPLC Mass Spectrometry -- 4.2 CZE Mass Spectrometry -- 4.3 Microchips -- 4.4 Nanospray -- 5 Identification of Posttranslational Modifications -- 5.1 Disulfide Bond Location -- 5.2 Other Posttranslational Modifications -- 6 Interpretation of Mass Spectrometric Data -- 6.1 Identification of Proteins by Database Search -- 6.2 De novo Sequencing of Peptides -- 7 References -- Section IV: Computer Sequence Analysis -- IV.1 Internet Resources for Protein Identification and Characterization -- 1 Introduction -- 2 General Approach -- 2.1 Experimental Attributes and Choice of Programs -- 2.2 Information in Databases -- 2.2.1 The Annotations in SWISS-PROT -- 2.2.2 SWISS-PROT Supplement TrEMBL -- 3 Identification and Characterization Tools at ExPASy -- 3.1 Identification Tools -- 3.1.1 Identification with Sequence Tags: TagIdent. , 3.1.2 Identification with Amino Acid Composition: AACompIdent.

Note: Intro -- Proteomics in Drug Research -- Contents -- A Personal Foreword -- Preface -- List of Contributors -- I Introduction -- 1 Administrative Optimization of Proteomics Networks for Drug Development -- 1.1 Introduction -- 1.2 Tasks and Aims of Administration -- 1.3 Networking -- 1.4 Evaluation of Biomarkers -- 1.5 A Network for Proteomics in Drug Development -- 1.6 Realization of Administrative Networking: the Brain Proteome Projects -- 1.6.1 National Genome Research Network: the Human Brain Proteome Project -- 1.6.2 Human Proteome Organisation: the Brain Proteome Project -- 1.6.2.1 The Pilot Phase -- References -- 2 Proteomic Data Standardization, Deposition and Exchange -- 2.1 Introduction -- 2.2 Protein Analysis Tools -- 2.2.1 UniProt -- 2.2.2 InterPro -- 2.2.3 Proteome Analysis -- 2.2.4 International Protein Index (IPI) -- 2.2.5 Reactome -- 2.3 Data Storage and Retrieval -- 2.4 The Proteome Standards Initiative -- 2.5 General Proteomics Standards (GPS) -- 2.6 Mass Spectrometry -- 2.7 Molecular Interactions -- 2.8 Summary -- References -- II Proteomic Technologies -- 3 Difference Gel Electrophoresis (DIGE): the Next Generation of Two-Dimensional Gel Electrophoresis for Clinical Research -- 3.1 Introduction -- 3.2 Difference Gel Electrophoresis: Next Generation of Protein Detection in 2-DE -- 3.2.1 Application of CyDye DIGE Minimal Fluors (Minimal Labeling with CyDye DIGE Minimal Fluors) -- 3.2.1.1 General Procedure -- 3.2.1.2 Example of Use: Identification of Kinetic Proteome Changes upon Ligand Activation of Trk-Receptors -- 3.2.2 Application of Saturation Labeling with CyDye DIGE Saturation Fluors -- 3.2.2.1 General Procedure -- 3.2.2.2 Example of Use: Analysis of 1000 Microdissected Cells from PanIN Grades for the Identification of a New Molecular Tumor Marker Using CyDye DIGE Saturation Fluors. , 3.2.3 Statistical Aspects of Applying DIGE Proteome Analysis -- 3.2.3.1 Calibration and Normalization of Protein Expression Data -- 3.2.3.2 Detection of Differentially Expressed Proteins -- 3.2.3.3 Sample Size Determination -- 3.2.3.4 Further Applications -- References -- 4 Biological Mass Spectrometry: Basics and Drug Discovery Related Approaches -- 4.1 Introduction -- 4.2 Ionization Principles -- 4.2.1 Matrix-Assisted Laser Desorption/Ionization (MALDI) -- 4.2.2 Electrospray Ionization -- 4.3 Mass Spectrometric Instrumentation -- 4.4 Protein Identification Strategies -- 4.5 Quantitative Mass Spectrometry for Comparative and Functional Proteomics -- 4.6 Metabolic Labeling Approaches -- 4.6.1 (15)N Labeling -- 4.6.2 Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) -- 4.7 Chemical Labeling Approaches -- 4.7.1 Chemical Isotope Labeling at the Protein Level -- 4.7.2 Stable Isotope Labeling at the Peptide Level -- 4.8 Quantitative MS for Deciphering Protein-Protein Interactions -- 4.9 Conclusions -- References -- 5 Multidimensional Column Liquid Chromatography (LC) in Proteomics - Where Are We Now? -- 5.1 Introduction -- 5.2 Why Do We Need MD-LC/MS Methods? -- 5.3 Basic Aspects of Developing a MD-LC/MS Method -- 5.3.1 General -- 5.3.2 Issues to be Considered -- 5.3.3 Sample Clean-up -- 5.3.4 Choice of Phase Systems in MD-LC -- 5.3.5 Operational Aspects -- 5.3.6 State-of-the-Art - Digestion Strategy Included -- 5.3.6.1 Multidimensional LC MS Approaches -- 5.4 Applications of MD-LC Separation in Proteomics - a Brief Survey -- 5.5 Sample Clean-Up: Ways to Overcome the "Bottleneck" in Proteome Analysis -- 5.6 Summary -- References -- 6 Peptidomics Technologies and Applications in Drug Research -- 6.1 Introduction -- 6.2 Peptides in Drug Research -- 6.2.1 History of Peptide Research -- 6.2.2 Brief Biochemistry of Peptides. , 6.2.3 Peptides as Drugs -- 6.2.4 Peptides as Biomarkers -- 6.2.5 Clinical Peptidomics -- 6.3 Development of Peptidomics Technologies -- 6.3.1 Evolution of Peptide Analytical Methods -- 6.3.2 Peptidomic Profiling -- 6.3.3 Top-Down Identification of Endogenous Peptides -- 6.4 Applications of Differential Display Peptidomics -- 6.4.1 Peptidomics in Drug Development -- 6.4.2 Peptidomics Applied to in vivo Models -- 6.5 Outlook -- References -- 7 Protein Biochips in the Proteomic Field -- 7.1 Introduction -- 7.2 Technological Aspects -- 7.2.1 Protein Immobilization and Surface Chemistry -- 7.2.2 Transfer and Detection of Proteins -- 7.2.3 Chip Content -- 7.3 Applications of Protein Biochips -- 7.4 Contribution to Pharmaceutical Research and Development -- References -- 8 Current Developments for the In Vitro Characterization of Protein Interactions -- 8.1 Introduction -- 8.2 The Model System: cAMP-Dependent Protein Kinase -- 8.3 Real-time Monitoring of Interactions Using SPR Biosensors -- 8.4 ITC in Drug Design -- 8.5 Fluorescence Polarization, a Tool for High-Throughput Screening -- 8.6 AlphaScreen as a Pharmaceutical Screening Tool -- 8.7 Conclusions -- References -- 9 Molecular Networks in Morphologically Intact Cells and Tissue-Challenge for Biology and Drug Development -- 9.1 Introduction -- 9.2 A Metaphor of the Cell -- 9.3 Mapping Molecular Networks as Patterns: Theoretical Considerations -- 9.4 Imaging Cycler Robots -- 9.5 Formalization of Network Motifs as Geometric Objects -- 9.6 Gain of Functional Information: Perspectives for Drug Development -- References -- III Applications -- 10 From Target to Lead Synthesis -- 10.1 Introduction -- 10.2 Materials and Methods -- 10.2.1 Cells and Culture Conditions -- 10.2.2 In Vitro Activity Testing -- 10.2.3 Affinity Chromatography -- 10.2.4 Electrophoresis and Protein Identification. , 10.2.5 BIAcore Analysis -- 10.2.6 Synthesis of Acyl Cyanides -- 10.2.6.1 Methyl 5-cyano-5-oxopentanoate -- 10.2.6.2 Methyl 6-cyano-6-oxohexanoate -- 10.2.6.3 Methyl-5-cyano-3-methyl-5-oxopentanoate -- 10.3 Results -- 10.4 Discussion -- References -- 11 Differential Phosphoproteome Analysis in Medical Research -- 11.1 Introduction -- 11.2 Phosphoproteomics of Human Platelets -- 11.2.1 Cortactin -- 11.2.2 Myosin Regulatory Light Chain -- 11.2.3 Protein Disulfide Isomerase -- 11.3 Identification of cAMP- and cGMP-Dependent Protein Kinase Substrates in Human Platelets -- 11.4 Identification of a New Therapeutic Target for Anti-Inflammatory Therapy by Analyzing Differences in the Phosphoproteome of Wild Type and Knock Out Mice -- 11.5 Concluding Remarks and Outlook -- References -- 12 Biomarker Discovery in Renal Cell Carcinoma Applying Proteome-Based Studies in Combination with Serology -- 12.1 Introduction -- 12.1.1 Renal Cell Carcinoma -- 12.2 Rational Approaches Used for Biomarker Discovery -- 12.3 Advantages of Different Proteome-Based Technologies for the Identification of Biomarkers -- 12.4 Type of Biomarker -- 12.5 Proteome Analysis of Renal Cell Carcinoma Cell Lines and Biopsies -- 12.6 Validation of Differentially Expressed Proteins -- 12.7 Conclusions -- References -- 13 Studies of Drug Resistance Using Organelle Proteomics -- 13.1 Introduction -- 13.1.1 The Clinical Problem and the Proteomics Response -- 13.2 Objectives and Experimental Design -- 13.2.1 The Cell Lines -- 13.2.2 Organelle Isolation -- 13.2.2.1 Criteria for Isolation -- 13.2.2.2 Plasma Membrane Isolation -- 13.2.3 Protein Fractionation and Identification -- 13.2.4 Quantitative Comparisons of Protein Abundances -- 13.3 Changes in Plasma Membrane and Nuclear Proteins in MCF-7 Cells Resistant to Mitoxantrone -- References. , 14 Clinical Neuroproteomics of Human Body Fluids: CSF and Blood Assays for Early and Differential Diagnosis of Dementia -- 14.1 Introduction -- 14.2 Neurochemical Markers of Alzheimer's Disease -- 14.2.1 β-Amyloid Precursor Protein (β-APP): Metabolism and Impact on AD Diagnosis -- 14.2.2 Tau Protein and its Phosphorylated Forms -- 14.2.2.1 Hyperphosphorylation of Tau as a Pathological Event -- 14.2.2.2 Phosphorylated Tau in CSF as a Biomarker of Alzheimer's Disease -- 14.2.3 Apolipoprotein E (ApoE) Genotype -- 14.2.4 Other Possible Factors -- 14.2.5 Combined Analysis of CSF Parameters -- 14.2.6 Perspectives: Novel Techniques to Search for AD Biomarkers - Mass Spectrometry (MS), Differential Gel Electrophoresis (DIGE), and Multiplexing -- 14.3 Conclusions -- References -- 15 Proteomics in Alzheimer's Disease -- 15.1 Introduction -- 15.2 Proteomic Analysis -- 15.2.1 Sample Preparation -- 15.2.2 Two-Dimensional Electrophoresis -- 15.2.3 Protein Quantification -- 15.2.4 Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectroscopy -- 15.3 Proteins with Deranged Levels and Modifications in AD -- 15.3.1 Synaptosomal Proteins -- 15.3.2 Guidance Proteins -- 15.3.3 Signal Transduction Proteins -- 15.3.4 Oxidized Proteins -- 15.3.5 Heat Shock Proteins -- 15.3.6 Proteins Enriched in Amyloid Plaques -- 15.4 Limitations -- References -- 16 Cardiac Proteomics -- 16.1 Heart Proteomics -- 16.1.1 Heart 2-D Protein Databases -- 16.1.2 Dilated Cardiomyopathy -- 16.1.3 Animal Models of Heart Disease -- 16.1.4 Subproteomics of the Heart -- 16.1.4.1 Mitochondria -- 16.1.4.2 PKC Signal Transduction Pathways -- 16.1.5 Proteomics of Cultured Cardiac Myocytes -- 16.1.6 Proteomic Characterization of Cardiac Antigens in Heart Disease and Transplantation -- 16.1.7 Markers of Acute Allograft Rejection -- 16.2 Vessel Proteomics -- 16.2.1 Proteomics of Intact Vessels. , 16.2.2 Proteomics of Isolated Vessel Cells.

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