Proteomic Profiling and Analytical Chemistry

The Crossroads

2nd Edition - March 2, 2016
Editors: Pawel Ciborowski, Jerzy Silberring
Language: English
Paperback ISBN:
9 7 8 - 0 - 4 4 4 - 6 3 6 8 8 - 1
eBook ISBN:
9 7 8 - 0 - 4 4 4 - 6 3 6 9 0 - 4

Proteomic Profiling and Analytical Chemistry: The Crossroads, Second Edition helps scientists without a strong background in analytical chemistry to understand principles of the mu… Read more

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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.

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

Pawel Ciborowski

Dr. Pawel Ciborowski obtained an MS in Biochemistry from Warsaw University and a PhD in Bacteriology/Biochemistry in 1983 from the National Institute of Hygiene in Warsaw. After spending two years as the Alexander von Humboldt Fellow at the University of Cologne and two years as Visiting Scientist at the University of Lund, he came to the University of Pittsburgh. Since 2003, he has been a faculty member at the University of Nebraska Medical Center where he conducts his own research and directs the Mass Spectrometry and Proteomics Core Facility and teaches courses in biochemistry, mass spectrometry, and proteomics. He is the author of over 70 papers and reviews, as well as several contributions to books and textbooks. His research is focused on the correlation of structure and function of proteins, their receptors and the impact of posttranslational modifications on their functions in understanding molecular mechanisms of pathological processes and applications of this information in designing new strategies for disease prevention, early diagnosis, and control. Using state-of-the-art techniques in proteomics, he is investigating the structure and function of proteins which are involved in (i) response to changes in cell's environment, (ii) response to stimuli such as tissue injury, infection, drug treatment, and (iii) during malignant transformation.

Affiliations and expertise

Mass Spectrometry and Proteomics Core Facility, University of Nebraska Medical Center, Omaha, NE, USA

Jerzy Silberring

Dr. Jerzy Silberring obtained a PhD in Chemistry in 1978 at Jagiellonian University, Kraków, Poland. He received a Docent of Biochemistry title at the University of Uppsala in 1991, and became a full professor of biochemistry at the Jagiellonian University in 2001. He spent 10 years at the Uppsala University and Karolinska Institute in Stockholm. He is the author of over 200 papers and reviews mainly on proteolytic enzymes, neuropeptides, neurobiology of pain and drug dependence, proteomics, and mass spectrometry. His investigations have contributed to our understanding of the regulation of neuropeptides and their fragments, discovery of novel peptides, and applications of mass spectrometry. He teaches biochemistry, neurobiology of behavior, and drug dependence at the AGH University of Science and Technology, Jagiellonian University, and the Centre of Polymer and Carbon Materials, Polish Academy of Sciences.

Affiliations and expertise

AGH University of Science and Technology, Kraków, Poland, and Centre of Polymer and Carbon Materials, Polish Academy of Sciences, Kraków, Poland

Life sciences

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