Thiol Redox Transitions in Cell Signaling, Part A (eBook)

Chemistry and Biochemistry of Low Molecular Weight and Protein Thiols

Enrique Cadenas, Lester Packer (Herausgeber)

eBook Download: EPUB

2010 | 1. Auflage
416 Seiten
Elsevier Science (Verlag)
978-0-12-381346-6 (ISBN)

Infos

This volume, along with its companion (volume 475), presents methods and protocols dealing with thiol oxidation-reduction reactions and their implications as they relate to cell signaling. This first installment of Cadenas and Packer's two-volume treatment specifically deals with glutathionylation and dethiolation, and peroxide removal by peroxiredoxins/thioredoxins and glutathione peroxidases. The critically acclaimed laboratory standard for 40 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Over 450 volumes have been published to date, and much of the material is relevant even today--truly an essential publication for researchers in all fields of life sciences.

*Along with companion volume, provides a full overview of techniques necessary to the study of thiol redox in relation to cell signaling
* Gathers tried and tested techniques from global labs, offering both new and tried-and-true methods
* Relevant background and reference information given for procedures can be used as a guide to developing protocols in a number of disciplines

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Thiol Redox Transitions in Cell Signaling, Part A, along with its companion (volume 475), presents methods and protocols dealing with thiol oxidation-reduction reactions and their implications as they relate to cell signaling. This first installment of Cadenas and Packer's two-volume treatment specifically deals with glutathionylation and dethiolation, and peroxide removal by peroxiredoxins/thioredoxins and glutathione peroxidases. The critically acclaimed laboratory standard for 40 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Over 450 volumes have been published to date, and much of the material is relevant even today--truly an essential publication for researchers in all fields of life sciences. - Along with companion volume, provides a full overview of techniques necessary to the study of thiol redox in relation to cell signaling- Gathers tried and tested techniques from global labs, offering both new and tried-and-true methods- Relevant background and reference information given for procedures can be used as a guide to developing protocols in a number of disciplines

Inhalt

Front Cover 1
Methods in Enzymology: Thiol Redox Transitions in Cell Signaling, Part A 4
Copyright Page 5
Contents 6
Contributors 12
Preface 20
Volumes in Series 22
Chapter 1: Changing Paradigms in Thiology: From Antioxidant Defense Toward Redox Regulation 50
1. Introduction 51
2. Early Concepts, Misconceptions, Unsettled Battles, and Persistent Confusion 52
3. The Discovery of "Natural" Free Radicals 55
4. Love Affairs Between Hydroperoxides and Thiolates or Selenolates 58
4.1. Glutathione peroxidases 58
4.2. Peroxiredoxins 63
4.3. Other thiol peroxidases 66
4.4. Redoxins 66
5. Toward Regulatory Circuits with Puzzle Stones from Redox Biochemistry 68
5.1. Triggering signals 69
5.2. Hydroperoxide sensors 70
5.3. Signal transducers 72
5.4. Targets 73
5.5. Shut-off switches and restoration of starting conditions 73
5.6. Signaling versus defense or modulation of redox signaling by competition 75
6. Conclusions and Perspectives 76
References 77
Chapter 2: Mass Spectrometry-Based Methods for the Determination of Sulfur and Related Metabolite Concentrations in Cell Extracts 90
1. Introduction 91
2. Analytical Methods for the Determination of Sulfur and Amino Acid Metabolites 93
3. Procedures 95
3.1. Cell growth and 15N metabolic labeling 95
3.2. Metabolite extraction 97
4. Absolute LC-ESI-MS/MS Quantification of Thiol and Amino Acid Metabolites in Yeast Extracts 98
4.1. Sample preparation 98
4.2. Liquid chromatography 99
4.3. Mass spectrometry 101
4.4. Data processing 103
4.5. Method validation 106
5. Qualitative and Quantitative Determination of Thiol and Amino Acid Metabolites in Yeast Extracts by Using an LTQ-Orbitrap Mass Spectrometer 113
5.1. Sample preparation 113
5.2. Direct introduction 113
5.3. LC/MS 113
5.4. Data processing 115
6. Discussion 117
7. Summary 119
Acknowledgments 122
References 122
Chapter 3: Use of Dimedone-Based Chemical Probes for Sulfenic Acid Detection: Evaluation of Conditions Affecting Probe Incorporation into Redox-Sensitive Proteins 126
1. Introduction 127
2. Materials 130
2.1. Solutions 130
2.2. Chemical modification agents 131
2.3. Proteins 132
3. Methods 132
3.1. Characterization of "DCP"-linked compounds 132
3.2. Protocols for labeling cysteine sulfenic acids within cellular proteins 135
4. Summary 141
Acknowledgments 141
References 142
Chapter 4: Use of Dimedone-Based Chemical Probes for Sulfenic Acid Detection: Methods to Visualize and Identify Labeled Proteins 144
1. Introduction 145
2. Biotin-Based Affinity Capture to Identify Proteins Containing Cysteine Sulfenic Acids 147
2.1. Materials 148
2.2. Methods 149
3. Detection Methods to Identify Oxidized Proteins and Cysteines 152
3.1. Targeted approaches: Western blot of affinity-enriched proteins to analyze proteins of interest 152
3.2. Targeted approaches: Immunoprecipitation of protein of interest followed by Western blot to detect biotin 153
3.3. Controls for endogenous biotinylation 155
3.4. Global approaches: Identification of overall sulfenic acid levels for cellular proteins in response to stimuli 155
3.5. Global approaches: Identification of oxidized proteins by mass spectrometry after biotin affinity capture 159
3.6. Identification of oxidized cysteine by MS–MS analysis 159
4. Summary 161
Acknowledgments 162
References 162
Chapter 5: Formation and Reactions of Sulfenic Acid in Human Serum Albumin 166
1. Introduction 167
2. Preparation of Albumin Solutions 168
2.1. Source of albumin for biochemical studies 168
2.2. Albumin delipidation 168
2.3. Albumin thiol reduction 169
2.4. Albumin quantification 169
2.5. Thiol quantification 169
2.6. Thiol blockage 172
3. Preparation of Oxidized Albumin 172
4. Detection of Albumin Sulfenic Acid 174
4.1. Sodium arsenite 175
4.2. 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) 175
4.3. Dimedone and mass spectrometry 176
4.4. Glutathione 179
5. Quantification of Albumin Sulfenic Acid Using Thionitrobenzoate (TNB) 180
6. Reactivity of Sulfenic Acid 181
7. Detection of Albumin Sulfinic Acid 183
8. Conclusions 183
Acknowledgments 183
References 184
Chapter 6: Determination of GSH, GSSG, and GSNO Using HPLC with Electrochemical Detection 186
1. Introduction 187
2. Methods 188
2.1. High-performance liquid chromatography with electrochemical detection 188
2.2. Hydrodynamic voltammogram of GSH, GSNO, and GSSG 189
2.3. GSNO detection in biological samples: Effect of sample preparation 191
2.4. Measurement of GSNO reductase activity using HPLC 193
3. Summary 194
Acknowledgments 195
References 195
Chapter 7: Measurement of Mixed Disulfides Including Glutathionylated Proteins 198
1. Introduction 199
2. Chemical Quantification of PSSX, PSSG, and PSSC 200
2.1. Principle 200
2.2. Reagents and solutions 201
2.3. Experimental procedure 201
2.4. Results 203
2.5. Typical data 203
2.6. Importance of basic pH 204
2.7. Importance of DTT and PSH 204
3. Visualization of PSSG by Western Blot 205
3.1. Principle 205
3.2. Methods and procedures 205
3.3. Typical results 206
Acknowledgments 206
References 207
Chapter 8: Detection and Quantification of Protein Disulfides in Biological Tissues: A Fluorescence-BasedProteomic Approach 210
1. Introduction 212
2. Material and Methods 213
2.1. Animals 213
2.2. Gel electrophoresis-based protein disulfide assay 213
2.3. Selection of the alkylating agents for measuring protein disulfide levels 214
2.4. Transformation of fluorescence units to nmoles of protein disulfide 216
2.5. 2D gel electrophoresis 216
2.6. Identification of proteins by MALDI-TOF/MS 217
3. Results 218
3.1. Measurement of changes in protein disulfide levels in response to oxidative stress 218
3.2. Measurement of changes in protein disulfide levels in young and old mice 218
3.3. Changes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity in young and old mice 220
4. Discussion 223
Acknowledgments 224
References 224
Chapter 9: Measurement and Identification of S-Glutathiolated Proteins 228
1. Introduction 229
2. Chemical Methods for the Measurement of Glutathiolated Proteins 231
2.1. Release of protein-bound GSH by oxidation 232
3. Detection of Glutathiolated Proteins by ESI/MS 233
3.1. Mass spectrometric analysis 234
4. Identification of S-Glutathiolated Proteins in Cells and Tissues 235
4.1. Western blot analysis 235
4.2. Immunohistochemical staining 240
4.3. Radioactive tagging 241
4.4. Biotin labeling 242
5 Conclusions 243
Acknowledgment 245
References 245
Chapter 10: Proteome Screens for Cys Residues Oxidation: The Redoxome 248
1. Introduction 249
2. General Considerations 250
2.1. Limits in the access to Cys-residues redox modifications 250
2.2. Acid quenching and Cys differential labeling 250
3. Overview of the Different Methods 251
3.1. 2DE-based methods 251
3.2. Shotgun proteomic: The MS-based ICAT technology 254
4. Results and Discussion 255
4.1. Methods 255
4.2. Results 259
5. Conclusions 263
Acknowledgments 263
References 263
Chapter 11: Identification by MS/MS of Disulfides Produced by a Functional Redox Transition 266
1. Introduction 267
2. MS/MS Identification of Redox-Switches in Protein 268
3. Analytical Procedure 269
3.1. Protein reduction and oxidation 269
3.2. Enzymatic digestions 270
3.3. LC–ESI-MS/MS 270
3.4. Data handling of mass spectra 270
3.5. Identification of the disulfide 270
4. Discussion 271
References 273
Chapter 12: Mass Spectrometry Approaches for the Redox Characterization of Protein Cysteine Residues: The Case of theTranscription Factor Pax-8 276
1. Introduction 277
2. Materials and Methods 279
2.1. Protein expression and functional analysis 279
2.2. Alkylation of protein samples with iodoacetamide and ESI-quadrupole-MS analysis 281
2.3. Enzymatic digestion and MALDI-TOF peptide-mapping experiments 281
3. Results 281
3.1. Combined functional and MS analysis of the Pax-8 Prd domain under various redox conditions 281
3.2. MS assignment of modified Cys residues in Pax-8 Prd domain 285
3.3. Functional role of the modified Cys residues on Pax-8 activity 288
4. Conclusions and Future Perspectives 290
Acknowledgments 292
References 292
Chapter 13: A Simple Method to Systematically Study Oxidatively Modified Proteins in Biological Samples and Its Applications 300
1. Introduction 301
2. Materials 303
2.1. Chemicals and other materials 303
3. Methods 304
4. Discussion 306
4.1. Advantages of the simple redox-based Cys-targeted proteomics method 306
4.2. Limitations of the redox-based Cys-targeted proteomics and alternative approaches 309
Acknowledgment 311
References 311
Chapter 14: Direct and Indirect Detection Methods for the Analysis of S-Nitrosylated Peptides and Proteins 314
1. Introduction 315
2. Indirect Detection of S-Nitrosylated Proteins: His-tag Switch 317
2.1. Protocol for the analysis of S-nitrosylation using the His-tag switch 318
3. Direct Analysis of S-Nitrosylation by MS 323
3.1. Protocol for the direct analysis of S-nitrosylated peptides 323
4. Conclusions 327
References 327
Chapter 15: A Rapid Approach for the Detection, Quantification, and Discovery of Novel Sulfenic Acid or S-Nitrosothiol Modified Proteins Using a Biotin-Switch Method 330
1. Introduction 331
1.1. Nitrosative protein oxidation 332
1.2. Sulfenic acid formation 337
2. Overview of Analytical Strategy 341
2.1. Generating S-nitrosocysteine 342
2.2. Tissue preparation 342
2.3. Detection of modified proteins using the biotin-switch method 343
2.4. Detection and purification of modified proteins after the biotin-switch method 343
3. Conclusions 345
Acknowledgments 346
References 346
Chapter 16: Protein Adducts of Aldehydic Lipid Peroxidation Products: Identification and Characterizationof Protein Adducts Using anAldehyde/Keto-Reactive Probein Combination with MassSpectrometry 354
1. Introduction 355
2. Modification of Proteins by Aldehydic Lipid Peroxidation Products 356
3. Redox Proteomics of Protein Targets of Reactive Lipid Peroxidation Products 357
4. Mass Spectrometry-Based Approaches for the Identification and Characterization of Protein Adducts of Aldehydic Lipid Peroxidation Products 359
5. Experimental Strategy of Using an Aldehyde/Keto-Reactive Probe for the Targeted Analysis of Protein Adducts of Aldehydic Lipid Peroxidation Products 360
5.1. ARP-labeling of aldehydic protein adducts of 2-alkenals and tryptic proteolysis 361
5.2. Enrichment of ARP-labeled peptide adducts using biotin avidin affinity chromatography 362
5.3. Tandem mass spectrometry for peptide identification and determining the site of adduction 363
5.4. Searching of MS/MS data against protein sequence databases for the identification of peptide sequences and peptide adducts 365
6. Applications of the ARP-Labeling Strategy 365
7. Interpretation of MS/MS Spectra of Protein Adducts and the Use of Diagnostic Marker Ions 372
8. Conclusion 373
Acknowledgment 376
References 377
Author Index 380
Subject Index 402
Color Plate 410

Produkt-Details

• Erscheint lt. Verlag: 22.7.2010
• Sprache: englisch
• Themenwelt: Studium ► 1. Studienabschnitt (Vorklinik) ► Physiologie
• Themenwelt: Naturwissenschaften ► Biologie ► Biochemie
• Themenwelt: Naturwissenschaften ► Biologie ► Zellbiologie
• Themenwelt: Naturwissenschaften ► Physik / Astronomie ► Angewandte Physik
• Themenwelt: Technik
• ISBN-10: 0-12-381346-8 / 0123813468
• ISBN-13: 978-0-12-381346-6 / 9780123813466