Proteomic Profiling and Analytical Chemistry

Proteomic Profiling and Analytical Chemistry: The Crossroads, Second Edition helps scientists without a strong background in analytical chemistry to understand principles of the multistep proteomic experiment necessary for its successful completion. It also helps researchers who do have an analytical chemistry background to break into the proteomics field. Highlighting points of junction between proteomics and analytical chemistry, this resource links experimental design with analytical measurements, data analysis, and quality control. This targeted point of view will help both biologists and chemists to better understand all components of a complex proteomic study.

The book provides detailed coverage of experimental aspects such as sample preparation, protein extraction and precipitation, gel electrophoresis, microarrays, dynamics of fluorescent dyes, and more. The key feature of this book is a direct link between multistep proteomic strategy and quality control routinely applied in analytical chemistry. This second edition features a new chapter on SWATH-MS, substantial updates to all chapters, including proteomic database search and analytical quantification, expanded discussion of post-hoc statistical tests, and additional content on validation in proteomics.

• Covers the analytical consequences of protein and peptide modifications that may have a profound effect on how and what researchers actually measure
• Includes practical examples illustrating the importance of problems in quantitation and validation of biomarkers
• Helps in designing and executing proteomic experiments with sound analytics

Analytical chemists, mass spectrometrists, researchers in proteomics, molecular biologists, biotechnologists, and pharmaceutical scientists

1. Introduction

• 1.1. Why Do Analytics Matter?
• 1.2. Expectations: Who and What?
• 1.3. What Is Next and Where Are We Going?

2. Biomolecules

• 2.1. Major Features and Characteristics of Proteins and Peptides
• 2.2. Hydrophilicity and Hydrophobicity
• 2.3. Effect of Protein Fragmentation
• 2.4. Effect of Posttranslational Modifications
• 2.5. Amino Acid Sequence and Separating Conditions
• 2.6. Cysteine and Methionine: Amino Acids Containing Sulfur
• 2.7. Protein Identification and Characterization
• 2.8. Structure–Function Relationship and Its Significance in Systems Biology Function
• 2.9. Protein Folding and Protein–Protein Interactions
• 2.10. Moonlighting of Proteins
• 2.11. Summary

3. General Strategies for Proteomic Sample Preparation

• 3.1. Introduction
• 3.2. Inhibitors of Proteolytic and Other Enzymes
• 3.3. Homogenization
• 3.4. Homogenization and Isolation of Organelles
• 3.5. Crude Protein Extraction
• 3.6. Serum and Cerebrospinal Fluid Protein Extraction
• 3.7. Fractionation Based on Size-Exclusion Filters
• 3.8. Chromatographic Methods of Protein Fractionation
• 3.9. Peptide Purification
• 3.10. Detergents, Lipids and DNA
• 3.11. Summary

4. Protein Extraction and Precipitation

• 4.1. Introduction
• 4.2. Focus on Hydrophobic Protein Extraction
• 4.3. The Role of Protein Solvation
• 4.4. Protein Precipitation
• 4.5. Salting Out
• 4.6. Isoelectric Point Precipitation
• 4.7. Organic Solvent-Driven Precipitation
• 4.8. Trichloroacetic Acid Precipitation

5. Online and Offline Sample Fractionation

• 5.1. Introduction
• 5.2. Strong Cation Exchange, Weak Cation Exchange, Continuous or Step Gradient?
• 5.3. Protein and Peptide Separation Based on Isoelectric Point
• 5.4. Capillary Columns for Proteomic Analyses

6. Immunoaffinity Depletion of Highly Abundant Proteins for Proteomic Sample Preparation

• 6.1. Introduction
• 6.2. Immunodepletion Techniques
• 6.3. Capacity of Immunodepletion Columns and Other Devices
• 6.4. Reproducibility
• 6.5. Quality Control of Immunodepletion
• 6.6. Albuminome
• 6.7. Summary

7. Gel Electrophoresis

• 7.1. Fundamentals of Gel Electrophoresis
• 7.2. Two-Dimensional Gel Electrophoresis

8. Quantitative Measurements in Proteomics: Mass Spectrometry

• 8.1. Introduction
• 8.2. Absolute Quantitation
• 8.3. Relative Quantitation in Proteomics
• 8.4. Summary

9. SWATH-MS: Data Acquisition and Analysis

• 9.1. Introduction
• 9.2. Tandem Mass Spectrometry for Quantitative Proteomics
• 9.3. SWATH-MS Data Acquisition
• 9.4. Overview of SWATH-MS Data Analysis
• 9.5. Summary

10. Top-Down Proteomics

• 10.1. Introduction
• 10.2. Protein Separation Methods
• 10.3. Mass Spectrometry of Intact Proteins
• 10.4. Software for Data Analysis

11. Proteomic Database Search and Analytical Quantification for Mass Spectrometry

• 11.1. Introduction
• 11.2. Protein Databases
• 11.3. Search Engines
• 11.4. Mass Spectrometry Data Searches: Things to Consider
• 11.5. Post-Database Search Data Processing
• 11.6. Searches for Posttranslational Modifications
• 11.7. Summary

12. Design and Statistical Analysis of Mass-Spectrometry-Based Quantitative Proteomics Data

• 12.1. Introduction
• 12.2. Mass Spectrometry-Based Quantitative Proteomics
• 12.3. Issues and Statistical Consideration on Experimental Design
• 12.4. Data Preprocessing for Statistical Analysis
• 12.5. Statistical Analysis of Protein Expression Data
• 12.6. Summary

13. Principles of Analytical Validation

• 13.1. Introduction
• 13.2. Liquid Chromatographic Methods
• 13.3. Validation of a Liquid Chromatographic Method: Identity, Assay, Impurities
• 13.4. Recovery
• 13.5. Accuracy
• 13.6. Precision
• 13.7. Calibration Curve, Linearity, and Sensitivity
• 13.8. Selectivity and Specificity
• 13.9. Stability
• 13.10. Aberrant Results and Errors in Analyses
• 13.11. Further Development of Methods Validation

14. Validation in Proteomics and Regulatory Affairs

• 14.1. The “Uphill Battle” of Validation
• 14.2. Accuracy and Precision
• 14.3. Experimental Design and Validation
• 14.4. Validation of the Method
• 14.5. Validation of Detection Levels
• 14.6. Validation of Reproducibility and Sample Loss
• 14.7. Validation of Performance of Instruments
• 14.8. Bioinformatics: Validation of Output of Proteomic Data
• 14.9. Cross-Validation of Initial Results
• 14.10. Proteomics and Regulatory Affairs