Amino Acids, Peptides and Proteins in Organic Chemistry Volume 5

Amino Acids, Peptides and Proteins in Organic Chemistry
 
Author:
Andrew B. Hughes
Publisher: Wiley-VCH
ISBN No: 978-3-527-63185-8
Release at: 2012
Pages: 510
Edition:
Volume 5 - Analysis and Function of Amino Acids and Peptides
File Size: 9 MB
File Type: pdf
Language: English



Content of Amino Acids, Peptides and Proteins in Organic Chemistry



1 Mass Spectrometry of Amino Acids and Proteins 1
Simin D. Maleknia and Richard Johnson
1.1 Introduction 1
1.1.1 Mass Terminology 1
1.1.2 Components of a Mass Spectrometer 4
1.1.3 Resolution and Mass Accuracy 6
1.1.4 Accurate Analysis of ESI Multiply Charged Ions 10
1.1.5 Fragment Ions 11
1.2 Basic Protein Chemistry and How it Relates to MS 21
1.2.1 Mass Properties of the Polypeptide Chain 21
1.2.2 In Vivo Protein Modifications 21
1.2.3 Ex Vivo Protein Modifications 26
1.3 Sample Preparation and Data Acquisition 28
1.3.1 Top-Down Versus Bottom-Up Proteomics 28
1.3.2 Shotgun Versus Targeted Proteomics 28
1.3.3 Enzymatic Digestion for Bottom-Up Proteomics 29
1.3.4 Liquid Chromatography and Capillary Electrophoresis for
Mixtures in Bottom-Up 30
1.4 Data Analysis of LC-MS/MS (or CE-MS/MS) of Mixtures 32
1.4.1 Identification of Proteins from MS/MS Spectra of Peptides 32
1.4.2 De Novo Sequencing 35
1.5 MS of Protein Structure, Folding, and Interactions 36
1.5.1 Methods to Mass-Tag Structural Features 37
1.6 Conclusions and Perspectives 40
References 40

2 X-Ray Structure Determination of Proteins and Peptides 51
Andrew J. Fisher
2.1 Introduction 51
2.1.1 Light Microscopy 51
2.1.2 X-Rays and Crystallography at the Start 52
2.1.3 X-Ray Crystallography Today 53
2.1.4 Limitations of X-Ray Crystallography 54
2.2 Growing Crystals 55
2.2.1 Why Crystals? 55
2.2.2 Basic Methods of Growing Protein Crystals 55
2.2.3 Protein Sample 59
2.2.4 Preliminary Crystal Analysis 59
2.2.5 Mounting Crystals for X-Ray Analysis 61
2.3 Symmetry and Space Groups 62
2.3.1 Crystals and the Unit Cell 62
2.3.2 Point Groups 65
2.3.3 Space Groups 66
2.3.4 Asymmetric Unit 67
2.4 X-Ray Scattering and Diffraction 67
2.4.1 X-Rays and Mathematical Representation of Waves 67
2.4.2 Interaction of X-Rays with Matter 70
2.4.3 Crystal Lattice, Miller Indices, and the Reciprocal Space 73
2.4.4 X-Ray Diffraction from a Crystal: Braggs Law 75
2.4.5 Braggs Law in Reciprocal Space 77
2.4.6 Fourier Transform Equation from a Lattice 79
2.4.7 Friedels Law and the Electron Density Equation 80
2.5 Collecting and Processing Diffraction Data 82
2.5.1 Data Collection Strategy 82
2.5.2 Symmetry and Scaling Data 83
2.6 Solving the Structure (Determining Phases) 83
2.6.1 Molecular Replacement 83
2.6.2 Isomorphous Replacement 85
2.6.3 MAD 88
2.7 Analyzing and Refining the Structure 90
2.7.1 Electron Density Interpretation and Model Building 90
2.7.2 Protein Structure Refinement 91
2.7.3 Protein Structure Validation 93
References 94

3 Nuclear Magnetic Resonance of Amino Acids, Peptides,
and Proteins 97
Andrea Bernini and Pierandrea Temussi
3.1 Introduction 97
3.1.1 Active Nuclei in NMR 98
3.1.2 Energy Levels and Spin States 98
3.1.3 Main NMR Parameters (Glossary) 99
3.1.3.1 Chemical Shift 99
3.1.3.2 Scalar Coupling Constants 100
3.1.3.3 NOE 100
3.1.3.4 RDC 101
3.2 Amino Acids 101
3.2.1 Historical Significance 101
3.2.2 Amino Acids Structure 101
3.2.3 Random Coil Chemical Shift 102
3.2.4 Spin Systems 105
3.2.5 Labile Protons 110
3.2.6 Contemporary Relevance: Metabolomics 112
3.3 Peptides 113
3.3.1 Historical Significance 113
3.3.2 Oligopeptides as Models for Conformational Transitions
in Proteins 114
3.3.3 Bioactive Peptides 116
3.3.4 Choice of the Solvent 117
3.3.4.1 Transport Fluids 118
3.3.4.2 Membranes 120
3.3.4.3 Receptor Cavities 122
3.3.5 Ensemble Calculations 125
3.3.6 Selected Examples from the Major Fields of Bioactive Peptides 125
3.3.6.1 Aspartame 125
3.3.6.2 Opioids 126
3.3.6.3 Transmembrane Helices 127
3.3.6.4 Cyclopeptides 128
3.4 Proteins 129
3.4.1 An Alternative to or a Validation of Diffractometric
Methods? 129
3.4.2 Protein Spectra 129
3.4.3 Wüthrichs Protocol 130
3.4.3.1 Sample Preparation 131
3.4.3.2 Recording NMR Spectra 131
3.4.3.3 Sequential Assignment 131
3.4.3.4 Conformational Constraints 132
3.4.3.5 Model Building 134
3.4.4 Recent Developments 134
3.4.5 Selected Structures 136
3.4.5.1 Superoxide Dismutases 137
3.4.5.2 Malate Synthase G 137
3.4.5.3 Interactions 138
3.5 Conclusions 145
References 146

4 Structure and Activity of N-Methylated Peptides 155
Raymond S. Norton
4.1 Introduction 155
4.2 Conformational Effects of N-Methylation 157
4.3 Effects of N-Methylation on Bioactive Peptides 159
4.3.1 Thyrotropin-Releasing Hormone 159
4.3.2 Cyclic Peptides 159
4.3.3 Somatostatin Analogs 160
4.3.4 Antimalarial Peptide 161
4.4 Concluding Remarks 162
References 163

5 High-Performance Liquid Chromatography of Peptides
and Proteins 167
Reinhard I. Boysen and Milton T.W. Hearn
5.1 Introduction 167
5.2 Basic Terms and Concepts in Chromatography 169
5.3 Chemical Structure of Peptides and Proteins 173
5.3.1 Biophysical Properties of Peptides and Proteins 173
5.3.2 Conformational Properties of Peptides and Proteins 176
5.3.3 Optical Properties of Peptides and Proteins 176
5.4 HPLC Separation Modes in Peptide and Protein
Analysis 177
5.4.1 SEC 178
5.4.2 RPC 179
5.4.3 NPC 181
5.4.4 HILIC 181
5.4.5 ANPC 183
5.4.6 HIC 184
5.4.7 IEX 187
5.4.8 AC 188
5.5 Method Development from Analytical to Preparative Scale
Illustrated for HP-RPC 189
5.5.1 Development of an Analytical Method 190
5.5.2 Scaling Up to Preparative Chromatography 196
5.5.3 Fractionation 198
5.5.4 Analysis of the Quality of the Fractionation 198
5.6 Multidimensional HPLC 198
5.6.1 Purification of Peptides and Proteins by MD-HPLC
Methods 200
5.6.2 Fractionation of Complex Peptide and Protein Mixtures
by MD-HPLC 202
5.6.3 Operational Strategies for MD-HPLC Methods 202
5.6.3.1 Off-line Coupling Mode for MD-HPLC Methods 202
5.6.3.2 On-Line Coupling Mode for MD-HPLC Methods 203
5.6.4 Design of an Effective MD-HPLC Scheme 203
5.6.4.1 Orthogonality of Chromatographic Modes 203
5.6.4.2 Compatibility Matrix of Chromatographic Modes 205
5.7 Conclusions 206
References 207

6 Local Surface Plasmon Resonance and Electrochemical Biosensing
Systems for Analyzing Functional Peptides 211
Masato Saito and Eiichi Tamiya
6.1 Localized Surface Plasmon Resonance (LSPR)-Based Microfluidics
Biosensor for the Detection of Insulin Peptide Hormone 211
6.1.1 LSPR and Micro Total Analysis Systems 211
6.1.2 Microfluidic LSPR Chip Fabrication and LSPR Measurement 212
6.1.3 Detection of the Insulin–Anti-Insulin Antibody Reaction
on a Chip 213
6.2 Electrochemical LSPR-Based Label-Free Detection of Melittin 215
6.2.1 Melittin and E-LSPR 215
6.2.2 Fabrication of E-LSPR Substrate and Formation of the Hybrid
Bilayer Membrane 215
6.2.3 Measurements of Membrane-Based Sensors for Peptide Toxin 217
6.3 Label-Free Electrochemical Monitoring of b-Amyloid (Ab)
Peptide Aggregation 218
6.3.1 Alzheimers Ab Aggregation and Electrochemical
Detection Method 218
6.3.2 Label-Free Electrochemical Detection of Ab Aggregation 219
References 221

7 Surface Plasmon Resonance Spectroscopy in the Biosciences 225
Jing Yuan, Yinqiu Wu, and Marie-Isabel Aguilar
7.1 Introduction 225
7.2 SPR-Based Optical Biosensors 225
7.3 Principle of Operation of SPR Biosensors 226
7.4 Description of a SPR Instrument 228
7.4.1 Sensor Surface 228
7.4.2 Flow System 229
7.4.3 Detection System 230
7.5 Application of SPR in Immunosensor Design 230
7.5.1 Assay Development 232
7.5.1.1 Immobilization of the Analyte to a Specific Chip Surface 232
7.5.1.2 Assay Design 233
7.6 Application of SPR in Membrane Interactions 234
7.6.1 General Protocols for Membrane Interaction
Studies by SPR 236
7.6.1.1 Liposome Preparation 236
7.6.1.2 Formation of Bilayer Systems 236
7.6.1.3 Analyte Binding to the Membrane System 237
7.6.1.4 Membrane Binding of Antimicrobial Peptides by SPR 238
7.7 Data Analysis 240
7.7.1 Linearization Analysis 240
7.7.2 Numerical Integration Analysis 241
7.7.3 Steady-State Approximations 242
7.8 Conclusions 243
References 244

8 Atomic Force Microscopy of Proteins 249
Adam Mechler
8.1 Foreword 249
8.1.1 Importance of Asking the Right Question 250
8.2 AFM 250
8.2.1 Principle and Basic Modes of Operation 250
8.2.2 How Does a Tip Tap? 251
8.3 Bioimaging Highlights 253
8.3.1 Protein Oligomerization, Aggregation, and Fibers 253
8.3.2 Membrane Binding and Lysis 255
8.3.3 Ion Channel Activity 257
8.3.4 Protein–DNA-Specific Binding 261
8.4 Issues 261
8.4.1 Resolution 262
8.4.2 Imaging Force 263
8.4.3 Repetitive Stress 264
8.4.4 Artifacts Related to too Low Free Amplitude 265
8.4.5 Transient Force and Bandwidth 266
8.4.6 Accuracy of Surface Tracking 266
8.4.7 Step Artifacts 268
8.5 Force Measurements 269
8.6 Liquid Imaging 269
8.7 Sample Preparation for Bioimaging 272
8.7.1 Adhesion 272
8.7.2 Physical Entrapment 273
8.7.3 Chemical Binding 274
8.8 Outlook 274
References 275

9 Solvent Interactions with Proteins and Other Macromolecules 277
Satoshi Ohtake, Yoshiko Kita, Kouhei Tsumoto, and Tsutomu Arakawa
9.1 Introduction 277
9.2 Solvent Applications 280
9.2.1 Research 280
9.2.2 Precipitation 287
9.2.3 Chromatography 288
9.2.4 Protein Refolding 296
9.2.5 Formulation 297
9.3 Solvent Application for Viruses 300
9.3.1 Isolation and Purification of Viruses 301
9.3.2 Stabilization and Formulation of Viruses 302
9.3.3 Inactivation of Viruses 309
9.4 Solvent Application for DNA 310
9.4.1 Isolation and Purification of DNA 310
9.4.2 Stability of DNA in a Cosolvent System 312
9.5 Mechanism 314
9.5.1 Physical Mechanism 315
9.5.1.1 Hydration 315
9.5.1.2 Excluded Volume 318
9.5.2 Thermodynamic Interaction 322
9.5.2.1 Group Interaction: Model Compound Solubility 322
9.5.3 Preferential Interaction 328
9.6 Protein–Solvent Interactions in Frozen and Freeze-Dried Systems 342
9.6.1 Frozen Systems 342
9.6.2 Freeze-Dried System 345
9.7 Conclusions 348
References 349

10 Role of Cysteine 361
Lalla A. Ba, Torsten Burkholz, Thomas Schneider, and Claus Jacob
10.1 Sulfur: A Redox Chameleon with Many Faces 361
10.2 Three Faces of Thiols: Nucleophilicity, Redox Activity, and
Metal Binding 365
10.3 Towards a Dynamic Picture of Disulfide Bonds 371
10.4 Chemical Protection and Regulation via S-Thiolation 374
10.5 ‘‘Dormant’’ Catalytic Sites 378
10.6 Peroxiredoxin/Sulfiredoxin Catalysis and Control Pathway 379
10.7 Higher Sulfur Oxidation States: From the Shadows to
the Heart of Biological Sulfur Chemistry 384
10.8 Cysteine as a Target for Oxidants, Metal Ions, and
Drug Molecules 388
10.9 Conclusions and Outlook 390
References 391

11 Role of Disulfide Bonds in Peptide and Protein Conformation 395
Keith K. Khoo and Raymond S. Norton
11.1 Introduction 395
11.2 Probing the Role of Disulfide Bonds 396
11.3 Contribution of Disulfide Bonds to Protein Stability 396
11.4 Role of Disulfide Bonds in Protein Folding 397
11.5 Role of Individual Disulfide Bonds in Protein Structure 399
11.6 Disulfide Bonds in Protein Dynamics 401
11.7 Disulfide Bonding Patterns and Protein Topology 403
11.7.1 Conservation and Evolution of Disulfide Bonding Patterns 403
11.7.2 Conservation of Disulfide Bonds 404
11.7.3 Cysteine Framework and Disulfide Connectivity 404
11.7.4 Non-Native Disulfide Connectivities 407
11.8 Applications 408
11.9 Conclusions 409
References 410

12 Quantitative Mass Spectrometry-Based Proteomics 419
Shao-En Ong
12.1 Introduction 419
12.2 Quantification in Biological MS 420
12.2.1 Label-Free Approaches in Quantitative MS Proteomics 423
12.2.2 SIL in Quantitative Proteomics 425
12.3 Identifying Proteins Interacting with Small Molecules with
Quantitative Proteomics 430
12.4 Conclusions 433
References 434

13 Two-Dimensional Gel Electrophoresis and Protein/Polypeptide
Assignment 439
Takashi Manabe and Ya Jin
13.1 Introduction 439
13.2 Aim of Protein Analysis and Development of 2-DE
Techniques 439
13.3 Current Status of 2-DE Techniques 441
13.3.1 Denaturing 2-DE for the Separation of Polypeptides 442
13.3.1.1 Principle 442
13.3.1.2 Procedures 444
13.3.1.3 Specific Features 445
13.3.2 Nondenaturing 2-DE for the Separation of Biologically Active
Proteins and Protein Complexes 445
13.3.2.1 Principle 445
13.3.2.2 Procedures 446
13.3.2.3 Specific Features 447
13.3.3 Blue-Native 2-DE for the Detection of Protein–Protein Interactions 448
13.3.3.1 Principle 448
13.3.3.2 Procedures 448
13.3.3.3 Specific Features 449
13.3.4 Visualization of Proteins Separated on 2-DE Gels 449
13.3.4.1 Fixing Before CBB, Silver, or Fluorescent Dye Staining 450
13.3.4.2 CBB Staining 450
13.3.4.3 Silver Staining 450
13.3.4.4 Reverse Staining with Zinc-Imidazole 451
13.3.4.5 Fluorescent Dye Staining 451
13.3.4.6 Quantitation 451
13.4 Development of Protein Assignment Techniques on 2-DE
Gels and Current Status of Mass Spectrometric Techniques 452
13.4.1 Development of Protein Assignment Techniques 452
13.4.2 MS-Based Assignment Techniques Utilizing Amino Acid
Sequence Databases 454
13.4.2.1 Sample Preparation for MS Analysis 455
13.4.2.2 MALDI-TOF-MS and PMF 456
13.4.2.3 MS/MS and Peptide Sequence Search 459
13.5 Conclusions 460
References 460

14 Bioinformatics Tools for Detecting Post-Translational Modifications
in Mass Spectrometry Data 463
Patricia M. Palagi, Erik Arhné, MarKus Müller, and Frédérique Lisacek
14.1 Introduction 463
14.2 PTM Discovery with MS 465
14.2.1 Detecting PTMs in MS and MS/MS Data 466
14.2.2 Discovering PTMs in MS or MS/MS Data 468
14.2.3 PTM Prediction Tools 469
14.2.3.1 From MS Data 469
14.2.3.2 From Sequence Data 469
14.3 Database Resources for PTM Analysis 470
14.4 Conclusions 473
References 473

Index 477

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