Amino Acids, Peptides and Proteins in Organic Chemistry:Volume 5 - Analysis and Function of Amino Acids and Peptides -- Contents -- List of Contributors -- 1 Mass Spectrometry of Amino Acids and Proteins -- 1.1 Introduction -- 1.1.1 Mass Terminology -- 1.1.2 Components of a Mass Spectrometer -- 1.1.3 Resolution and Mass Accuracy -- 1.1.4 Accurate Analysis of ESI Multiply Charged Ions -- 1.1.5 Fragment Ions -- 1.2 Basic Protein Chemistry and How it Relates to MS -- 1.2.1 Mass Properties of the Polypeptide Chain -- 1.2.2 In Vivo Protein Modi.cations -- 1.2.3 Ex Vivo Protein Modi.cations -- 1.3 Sample Preparation and Data Acquisition -- 1.3.1 Top-Down Versus Bottom-Up Proteomics -- 1.3.2 Shotgun Versus Targeted Proteomics -- 1.3.3 Enzymatic Digestion for Bottom-Up Proteomics -- 1.3.4 Liquid Chromatography and Capillary Electrophoresis for Mixtures in Bottom-Up -- 1.4 Data Analysis of LC-MS/MS (or CE-MS/MS) of Mixtures -- 1.4.1 Identi.cation of Proteins from MS/MS Spectra of Peptides -- 1.4.2 De Novo Sequencing -- 1.5 MS of Protein Structure, Folding, and Interactions -- 1.5.1 Methods to Mass-Tag Structural Features -- 1.6 Conclusions and Perspectives -- References -- 2 X-Ray Structure Determination of Proteins and Peptides -- 2.1 Introduction -- 2.1.1 Light Microscopy -- 2.1.2 X-Rays and Crystallography at the Start -- 2.1.3 X-Ray Crystallography Today -- 2.1.4 Limitations of X-Ray Crystallography -- 2.2 Growing Crystals -- 2.2.1 Why Crystals? -- 2.2.2 Basic Methods of Growing Protein Crystals -- 2.2.3 Protein Sample -- 2.2.4 Preliminary Crystal Analysis -- 2.2.5 Mounting Crystals for X-Ray Analysis -- 2.3 Symmetry and Space Groups -- 2.3.1 Crystals and the Unit Cell -- 2.3.2 Point Groups -- 2.3.3 Space Groups -- 2.3.4 Asymmetric Unit -- 2.4 X-Ray Scattering and Diffraction -- 2.4.1 X-Rays and Mathematical Representation of Waves.

2.4.2 Interaction of X-Rays with Matter -- 2.4.3 Crystal Lattice, Miller Indices, and the Reciprocal Space -- 2.4.4 X-Ray Diffraction from a Crystal: Bragg.s Law -- 2.4.5 Bragg.s Law in Reciprocal Space -- 2.4.6 Fourier Transform Equation from a Lattice -- 2.4.7 Friedel' s Law and the Electron Density Equation -- 2.5 Collecting and Processing Diffraction Data -- 2.5.1 Data Collection Strategy -- 2.5.2 Symmetry and Scaling Data -- 2.6 Solving the Structure (Determining Phases) -- 2.6.1 Molecular Replacement -- 2.6.2 Isomorphous Replacement -- 2.6.3 MAD -- 2.7 Analyzing and Re.ning the Structure -- 2.7.1 Electron Density Interpretation and Model Building -- 2.7.2 Protein Structure Refinement -- 2.7.3 Protein Structure Validation -- References -- 3 Nuclear Magnetic Resonance of Amino Acids, Peptides, and Proteins -- 3.1 Introduction -- 3.1.1 Active Nuclei in NMR -- 3.1.2 Energy Levels and Spin States -- 3.1.3 Main NMR Parameters (Glossary) -- 3.1.3.1 Chemical Shift -- 3.1.3.2 Scalar Coupling Constants -- 3.1.3.3 NOE -- 3.1.3.4 RDC -- 3.2 Amino Acids -- 3.2.1 Historical Significance -- 3.2.2 Amino Acids Structure -- 3.2.3 Random Coil Chemical Shift -- 3.2.4 Spin Systems -- 3.2.5 Labile Protons -- 3.2.6 Contemporary Relevance: Metabolomics -- 3.3 Peptides -- 3.3.1 Historical Significance -- 3.3.2 Oligopeptides as Models for Conformational Transitions in Proteins -- 3.3.3 Bioactive Peptides -- 3.3.4 Choice of the Solvent -- 3.3.4.1 Transport Fluids -- 3.3.4.2 Membranes -- 3.3.4.3 Receptor Cavities -- 3.3.5 Ensemble Calculations -- 3.3.6 Selected Examples from the Major Fields of Bioactive Peptides -- 3.3.6.1 Aspartame -- 3.3.6.2 Opioids -- 3.3.6.3 Transmembrane Helices -- 3.3.6.4 Cyclopeptides -- 3.4 Proteins -- 3.4.1 An Alternative to or a Validation of Diffractometric Methods? -- 3.4.2 Protein Spectra -- 3.4.3 Wüthrich.s Protocol.

3.4.3.1 Sample Preparation -- 3.4.3.2 Recording NMR Spectra -- 3.4.3.3 Sequential Assignment -- 3.4.3.4 Conformational Constraints -- 3.4.3.5 Model Building -- 3.4.4 Recent Developments -- 3.4.5 Selected Structures -- 3.4.5.1 Superoxide Dismutases -- 3.4.5.2 Malate Synthase G -- 3.4.5.3 Interactions -- 3.5 Conclusions -- References -- 4 Structure and Activity of N-Methylated Peptides -- 4.1 Introduction -- 4.2 Conformational Effects of N-Methylation -- 4.3 Effects of N-Methylation on Bioactive Peptides -- 4.3.1 Thyrotropin-Releasing Hormone -- 4.3.2 Cyclic Peptides -- 4.3.3 Somatostatin Analogs -- 4.3.4 Antimalarial Peptide -- 4.4 Concluding Remarks -- References -- 5 High-Performance Liquid Chromatography of Peptides and Proteins -- 5.1 Introduction -- 5.2 Basic Terms and Concepts in Chromatography -- 5.3 Chemical Structure of Peptides and Proteins -- 5.3.1 Biophysical Properties of Peptides and Proteins -- 5.3.2 Conformational Properties of Peptides and Proteins -- 5.3.3 Optical Properties of Peptides and Proteins -- 5.4 HPLC Separation Modes in Peptide and Protein Analysis -- 5.4.1 SEC -- 5.4.2 RPC -- 5.4.3 NPC -- 5.4.4 HILIC -- 5.4.5 ANPC -- 5.4.6 HIC -- 5.4.7 IEX -- 5.4.8 AC -- 5.5 Method Development from Analytical to Preparative Scale Illustrated for HP-RPC -- 5.5.1 Development of an Analytical Method -- 5.5.2 Scaling Up to Preparative Chromatography -- 5.5.3 Fractionation -- 5.5.4 Analysis of the Quality of the Fractionation -- 5.6 Multidimensional HPLC -- 5.6.1 Puri.cation of Peptides and Proteins by MD-HPLC Methods -- 5.6.2 Fractionation of Complex Peptide and Protein Mixtures by MD-HPLC -- 5.6.3 Operational Strategies for MD-HPLC Methods -- 5.6.3.1 Off-line Coupling Mode for MD-HPLC Methods -- 5.6.3.2 On-Line Coupling Mode for MD-HPLC Methods -- 5.6.4 Design of an Effective MD-HPLC Scheme -- 5.6.4.1 Orthogonality of Chromatographic Modes.

5.6.4.2 Compatibility Matrix of Chromatographic Modes -- 5.7 Conclusions -- References -- 6 Local Surface Plasmon Resonance and Electrochemical Biosensing Systems for Analyzing Functional Peptides -- 6.1 Localized Surface Plasmon Resonance (LSPR)-Based Microfluidics Biosensor for the Detection of Insulin Peptide Hormone -- 6.1.1 LSPR and Micro Total Analysis Systems -- 6.1.2 Microfluidic LSPR Chip Fabrication and LSPR Measurement -- 6.1.3 Detection of the Insulin-Anti-Insulin Antibody Reaction on a Chip -- 6.2 Electrochemical LSPR-Based Label-Free Detection of Melittin -- 6.2.1 Melittin and E-LSPR -- 6.2.2 Fabrication of E-LSPR Substrate and Formation of the Hybrid Bilayer Membrane -- 6.2.3 Measurements of Membrane-Based Sensors for Peptide Toxin -- 6.3 Label-Free Electrochemical Monitoring of b-Amyloid (Ab) Peptide Aggregation -- 6.3.1 Alzheimer.s Ab Aggregation and Electrochemical Detection Method -- 6.3.2 Label-Free Electrochemical Detection of Ab Aggregation -- References -- 7 Surface Plasmon Resonance Spectroscopy in the Biosciences -- 7.1 Introduction -- 7.2 SPR-Based Optical Biosensors -- 7.3 Principle of Operation of SPR Biosensors -- 7.4 Description of a SPR Instrument -- 7.4.1 Sensor Surface -- 7.4.2 Flow System -- 7.4.3 Detection System -- 7.5 Application of SPR in Immunosensor Design -- 7.5.1 Assay Development -- 7.5.1.1 Immobilization of the Analyte to a Speci.c Chip Surface -- 7.5.1.2 Assay Design -- 7.6 Application of SPR in Membrane Interactions -- 7.6.1 General Protocols for Membrane Interaction Studies by SPR -- 7.6.1.1 Liposome Preparation -- 7.6.1.2 Formation of Bilayer Systems -- 7.6.1.3 Analyte Binding to the Membrane System -- 7.6.1.4 Membrane Binding of Antimicrobial Peptides by SPR -- 7.7 Data Analysis -- 7.7.1 Linearization Analysis -- 7.7.2 Numerical Integration Analysis -- 7.7.3 Steady-State Approximations.

7.8 Conclusions -- References -- 8 Atomic Force Microscopy of Proteins -- 8.1 Foreword -- 8.1.1 Importance of Asking the Right Question -- 8.2 AFM -- 8.2.1 Principle and Basic Modes of Operation -- 8.2.2 How Does a Tip Tap? -- 8.3 Bioimaging Highlights -- 8.3.1 Protein Oligomerization, Aggregation, and Fibers -- 8.3.2 Membrane Binding and Lysis -- 8.3.3 Ion Channel Activity -- 8.3.4 Protein-DNA-Specific Binding -- 8.4 Issues -- 8.4.1 Resolution -- 8.4.2 Imaging Force -- 8.4.3 Repetitive Stress -- 8.4.4 Artifacts Related to too Low Free Amplitude -- 8.4.5 Transient Force and Bandwidth -- 8.4.6 Accuracy of Surface Tracking -- 8.4.7 Step Artifacts -- 8.5 Force Measurements -- 8.6 Liquid Imaging -- 8.7 Sample Preparation for Bioimaging -- 8.7.1 Adhesion -- 8.7.2 Physical Entrapment -- 8.7.3 Chemical Binding -- 8.8 Outlook -- References -- 9 Solvent Interactions with Proteins and Other Macromolecules -- 9.1 Introduction -- 9.2 Solvent Applications -- 9.2.1 Research -- 9.2.2 Precipitation -- 9.2.3 Chromatography -- 9.2.4 Protein Refolding -- 9.2.5 Formulation -- 9.3 Solvent Application for Viruses -- 9.3.1 Isolation and Puri.cation of Viruses -- 9.3.2 Stabilization and Formulation of Viruses -- 9.3.3 Inactivation of Viruses -- 9.4 Solvent Application for DNA -- 9.4.1 Isolation and Puri.cation of DNA -- 9.4.2 Stability of DNA in a Cosolvent System -- 9.5 Mechanism -- 9.5.1 Physical Mechanism -- 9.5.1.1 Hydration -- 9.5.1.2 Excluded Volume -- 9.5.2 Thermodynamic Interaction -- 9.5.2.1 Group Interaction: Model Compound Solubility -- 9.5.3 Preferential Interaction -- 9.6 Protein-Solvent Interactions in Frozen and Freeze-Dried Systems -- 9.6.1 Frozen Systems -- 9.6.2 Freeze-Dried System -- 9.7 Conclusions -- References -- 10 Role of Cysteine -- 10.1 Sulfur: A Redox Chameleon with Many Faces.

10.2 Three Faces of Thiols: Nucleophilicity, Redox Activity, and Metal Binding.