Proteomic Profiling and Analytical Chemistry

Proteomic Profiling and Analytical Chemistry helps scientists without a strong background in analytical chemistry to understand basic analytical principles and apply them to proteomics profiling. In most proteomic profiling experiments, liquid chromatography is used; this method is also used widely in analytical chemistry. This book bridges the gap between overly specialized courses and books in mass spectrometry, proteomics and analytical chemistry. It also helps researchers with an analytical chemistry background to break into the proteomics field.

Proteomic Profiling and Analytical Chemistry focuses on practical applications for proteomic research helping readers to design better experiments and to more easily interpret, analyze and validate the resulting data. Experimental aspects such as sample preparation, protein extraction and precipitation, gel electrophoresis, microarrays, dynamics of fluorescent dyes, and more are all covered in detail.

• 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.

Preface

1. Introduction

1.1 Why 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 Post-translational 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

References

3. Fundamental Strategies of Protein and Peptide Sample Preparation

3.1 General Strategies for Proteomic Sample Fractionation

3.2 Capillary Columns for Proteomic Analyses

3.3 Ion-Exchange Chromatography

3.4 Protein and Peptide Separation Based on Isoelectric Point

4. Protein Extraction and Precipitation

4.1 Introduction

4.2 Inhibitors of Proteolytic and Other Enzymes

4.3 Focus on Hydrophobic Protein Extraction

4.4 On the Role of Protein Solvation

4.5 Protein Precipitation

4.6 Salting Out

4.7 Isoelectric Point Precipitation

4.8 Organic Solvent-Driven Precipitation

4.9 Trichloroacetic Acid Precipitation

4.10 Detergents, Lipids, and Final Remarks

References

5. Immunoaffinity Depletion of High-Abundant Proteins for Proteomic Sample Preparation

5.1 Capacity of Immunodepletion Columns and Other Devices

5.2 Reproducibility

5.3 Quality Control of Immunodepletion

5.4 Albuminome

5.5 Summary

References

6. Gel Electrophoresis

6.1 Fundamentals of Gel Electrophoresis

6.2 Two-Dimensional Gel Electrophoresis

7. Quantitative Measurements in Proteomics

7.1 Introduction

7.2 Absolute Quantitation

7.3 Relative Quantitation in Proteomics

7.4 Summary

Acknowledgments

References

8. Proteomic Database Search and Analytical Quantification

8.1 Introduction

8.2 Search Engines

8.3 Protein Databases

8.4 Ionization Mode and Mass Spectrometry Platform

8.5 Search Parameters—Mass Tolerance

8.6 Miscleavage: Friend or Foe?

8.7 Searches for Post-translational Modifications

8.8 Summary

References

9. Microarrays and Dynamics of Fluorescent Dyes

9.1 Historical Perspective

9.2 Protein Arrays, a Variety of Detection Approaches

9.3 Fluorescence Labeling

9.4 Closing Remarks

References

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

10.1 Introduction

10.2 Mass Spectrometry-Based Quantitative Proteomics

10.3 Issues and Statistical Consideration on Experimental Design

10.4 Sample Size Calculation

10.5 Statistical Analysis

10.6 Comparing Protein Expressions between Groups

10.7 Summary

References

11. Principles of Analytical Validation

11.1 Introduction

11.2 Validation of a High-Performance Liquid Chromatographic (HPLC) Method: Identity, Assay, Impurities

11.3 Recovery

11.4 Accuracy

11.5 Precision

11.6 Calibration Curve, Linearity, and Sensitivity

11.7 Selectivity and Specificity

11.8 Stability

11.9 Aberrant Results and Errors in Analyses

11.10 Quantitative Western Blot Analysis and ELISA

11.11 Further Development of Methods Validation

References

12. Validation in Proteomics and Regulatory Affairs

12.1 The “Uphill Battle” of Validation

12.2 Accuracy and Precision

12.3 Experimental Design and Validation

12.4 Validation of the Method

12.5 Validation of Detection Levels

12.6 Validation of Reproducibility and Sample Loss

12.7 Validation of Performance of Instruments

12.8 Bioinformatics: Validation of an Output of Proteomic Data

12.9 Proteomics and Regulatory Affairs

References

Index