Proteomics in Biology, Part B, Volume 586

Chapter One: Protein Cysteinyl-S-Nitrosylation: Analysis and Quantification

• Abstract
• 1 Introduction
• 2 SNOFlo Methodology
• 3 Applications

Chapter Two: Quantitative Proteomics of the E. coli Membranome

• Abstract
• 1 Introduction—Pipeline Overview
• 2 Sample Preparation
• 3 Peptide/Protein Identification
• 4 Quantification
• 5 Functional Annotation
• 6 Conclusion
• Acknowledgments

Chapter Three: Comparative Analysis and Validation of Different Steps in Glycomics Studies

• Abstract
• 1 Introduction
• 2 Experimental Design and Randomization
• 3 Sample Preparation for UPLC N-Glycan Analysis
• 4 Robustness of the Sample Preparation Method
• 5 Analysis of Sources of Variation
• 6 Method Validation (Between-Day and Between-Analyst Variation)
• 7 UPLC Analysis of 2-AB-Labeled N-Glycans
• 8 Data Analysis and Quality Control
• 9 Summary and Conclusions
• Acknowledgments

Chapter Four: Analysis of Translocation-Competent Secretory Proteins by HDX-MS

• Abstract
• 1 Introduction
• 2 Preparation of Polypeptides
• 3 Isotope Labeling
• 4 Mass Spectrometric Analysis
• 5 Data Visualization and Interpretation
• 6 Identifying Statistically Significant Conformational Differences in Comparative Local HDX-MS
• 7 Conclusions
• Acknowledgments

Chapter Five: Site-Specific Quantification of Lysine Acetylation Using Isotopic Labeling

• Abstract
• 1 Introduction
• 2 Principle of Site-Specific Quantification of Lysine Acetylation Using Isotopic Labeling
• 3 Experimental Procedures
• 4 Conclusion

Chapter Six: Posttranslational Modifications and Plant–Environment Interaction

• Abstract
• 1 Introduction
• 2 Phosphorylation-Dependent Signal Transduction
• 3 Glycosylation and Protein Transport
• 4 Ubiquitination and Sumoylation-Dependent Protein Regulation
• 5 Role of Oxidative Modifications in Energy Production
• 6 Acetylation and Plant Immune Response
• 7 Posttranslational Modifications and Protein–Protein Interaction
• 8 Conclusion

Chapter Seven: Identification of Posttranslational Modifications of Endogenous Chromatin Proteins From Testicular Cells by Mass Spectrometry

• Abstract
• 1 Introduction
• 2 Isolation of Different Stages of Rat Testicular Germ Cells
• 3 Extraction of Nuclear Basic Proteins
• 4 Purification of Chromatin Proteins by RP-HPLC
• 5 Mass Spectrometry Analysis
• 6 Future Perspectives and Challenges
• 7 Biological Implications in Spermatogenesis
• Acknowledgments

Chapter Eight: Quantitative Analysis of Yeast Checkpoint Protein Kinase Activity by Combined Mass Spectrometry Enzyme Assays

• Abstract
• 1 Introduction
• 2 Established Methods to Monitor Rad53 Activation
• 3 A Direct Quantitative Immunoprecipitation Kinase Activity Assay for Endogenous Rad53
• 4 Mass Spectrometric Analysis of Rad53 Autophosphorylation
• 5 Conclusions

Chapter Nine: Retrieving Quantitative Information of Histone PTMs by Mass Spectrometry

• Abstract
• 1 Introduction
• 2 MS-Based Proteomics
• 3 Analysis of Histone Modifications: Methods and Strategies
• 4 Clinical Applications
• 5 Conclusion

Chapter Ten: Evaluating Exosome Protein Content Changes Induced by Virus Activity Using SILAC Labeling and LC-MS/MS

• Abstract
• 1 Introduction
• 2 Experimental Design
• 3 Detailed Protocol
• 4 Conclusions

Chapter Eleven: Exoproteomics of Pathogens: Analysis of Toxins and Other Virulence Factors by Proteomics

• Abstract
• 1 Introduction
• 2 Exoproteome Sample Preparation and Fractionation
• 3 Shotgun Mass Spectrometry for Discovery of Toxins and Other Virulence Factors
• 4 Data Interpretation
• 5 Examples of Specific Applications
• 6 Conclusion

Chapter Twelve: Integrated and Quantitative Proteomics of Human Tumors

• Abstract
• 1 Introduction
• 2 Equipment, Material, and Buffers
• 3 Wet Lab Protocol
• 4 Dry Lab Protocol
• 5 An Example
• 6 Conclusion
• Acknowledgments

Chapter Thirteen: Mass Spectrometry-Based Analysis for the Discovery and Validation of Potential Colorectal Cancer Stool Biomarkers

• Abstract
• 1 Introduction
• 2 Proteomics: From Discovery to Translation
• 3 Sample Preparation
• 4 Proteomic Discovery Protocols
• 5 Protein Quantitation Protocols
• 6 Future Directions

Chapter Fourteen: Mass Spectrometry-Based Methodology for Identification of Native Histone Variant Modifications From Mammalian Tissues and Solid Tumors

• Abstract
• 1 Introduction
• 2 Mass Spectrometry-Based Methods Overview
• 3 Protocol
• 4 Notes
• 5 Concluding Remarks
• Acknowledgments

Chapter Fifteen: Drug Target Identification Using an iTRAQ-Based Quantitative Chemical Proteomics Approach—Based on a Target Profiling Study of Andrographolide

• Abstract
• 1 Introduction and General Principles
• 2 Materials, Equipment, and Solutions
• 3 Protocol
• 4 Discussion
• Acknowledgments

Chapter Sixteen: A Super-SILAC Strategy for the Accurate and Multiplexed Profiling of Histone Posttranslational Modifications

• Abstract
• 1 Introduction
• 2 Overview of the Method
• 3 Material
• 4 Methods
• 5 Conclusions
• Acknowledgments

Chapter Seventeen: A Comparison of Two-Hybrid Approaches for Detecting Protein–Protein Interactions

• Abstract
• 1 Introduction
• 2 The Bacterial Two-Hybrid System
• 3 The Yeast Two-Hybrid System
• 4 Comparison of Yeast and Bacterial Two-Hybrid Methods
• 5 Conclusion

Chapter Eighteen: Recent Achievements in Characterizing the Histone Code and Approaches to Integrating Epigenomics and Systems Biology

• Abstract
• 1 Introduction
• 2 Sample Preparation
• 3 MS-Based Strategies for Histone Analysis
• 4 Future Perspective/Outlook
• Acknowledgments

Chapter Nineteen: Rapid Proteomics to Prospect and Validate Novel Bacterial Metabolism Induced by Environmental Burden

• Abstract
• 1 Introduction
• 2 Method Summary
• 3 Stable Isotope Dimethyl-Labeling Protocol
• 4 Applications
• 5 Conclusion
• Acknowledgments

Chapter Twenty: Quantitation of Human Metallothionein Isoforms in Cells, Tissues, and Cerebrospinal Fluid by Mass Spectrometry

• Abstract
• 1 Introduction
• 2 Methodology
• 3 Conclusions
• Acknowledgments

Chapter Twenty-One: A Cautionary Tale on the Inclusion of Variable Posttranslational Modifications in Database-Dependent Searches of Mass Spectrometry Data

• Abstract
• 1 Introduction
• 2 Scores and Thresholds
• 3 The Power of Calculating False Discovery Rates
• 4 The Advantages and Disadvantages of Including Some Common Posttranslational Modifications as Variable Modifications in Database-Dependent Searches
• 5 The Special Case of the GlyGly Modification
• 6 Conclusions
• Acknowledgments

Chapter Twenty-Two: Kinase Assay-Linked Phosphoproteomics: Discovery of Direct Kinase Substrates

• Abstract
• 1 Introduction
• 2 Overview of Kinase Substrate Identification Methods
• 3 Kinase Assay-Linked Phosphoproteomics Approach
• 4 Conclusions
• 5 Materials
• Acknowledgments

Proteomics in Biology, Part B, the latest volume in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods in proteomics.

• Continues the legacy of this premier serial with quality chapters that focus on proteomics
• Contains contributions from leading authorities

Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists