Proteomics in Biology, Part B

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