Proteomic Profiling and Analytical Chemistry

Proteomic Profiling and Analytical Chemistry

The Crossroads

1st Edition - December 31, 2012

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  • Editors: Pawel Ciborowski, Jerzy Silberring
  • eBook ISBN: 9780444593917

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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.

Key Features

  • 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.

Table of Contents

  • 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


    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


    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


    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



    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


    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


    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


    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


    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



Product details

  • No. of pages: 264
  • Language: English
  • Copyright: © Elsevier 2013
  • Published: December 31, 2012
  • Imprint: Elsevier
  • eBook ISBN: 9780444593917

About the Editors

Pawel Ciborowski

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

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

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