Catalyzing the Future of Proteomics: Protein Separation

Published by: BioInformatics, LLC

Published: Dec. 1, 2001 - 102 Pages

Table of Contents

Executive Overview

• Key Findings

Introduction

• Protein Separation for Proteomics
  
• Isoelectric Focusing
  
• Polyacrylamide Gel Electrophoresis
  
• Two-Dimensional Gel Electrophoresis
  
• Liquid Chromatography
  
• Capillary Electrophoresis
  
• Separation Technology Innovations

Section 2. Study Methodology and Demographics

Study Methodology

• Objectives
  
• Comments

Demographics

• Market Segment
  
• Organization Size
  
• Laboratory Size
  
• Region
  
• Job Position
  
• Area(s) of Research

Section 3. Significant Findings

Isoelectric Focusing (IEF)

• Current use of IEF
  
• Types of IEF employed for specific separation techniques
  
• Number of one-dimensional IEF experiments performed per week
  
• pH range used for one- and two-dimensional IEF applications
  
• Sequential running IEF gel use

Polyacrylamide Gel Electrophoresis (PAGE)

• PAGE use
  
• One-dimensional PAGE use
  
• Number of one-dimensional PAGE gels typically run per week
  
• Types of PAGE gels employed for specific separation techniques
  
• Percentage of PAGE resolving gels typically employed in one- or two-dimensional applications

Two-Dimensional Gel Electrophoresis (2DGE)

• 2DGE use
  
• Number of 2DGE experiments typically performed per week
  
• Problems of greatest concern when using 2DGE
  
• Current and future use of separation techniques other than 2DGE
  
• Current and future use of a combination of separation techniques
  
• Most influential factors in deciding to use a separation technique other than 2DGE

Technologies & Suppliers

• Top-of-mind company for products and services for protein separation
  
• Applications for which specific separation techniques are used
  
• Degree of sample purification required for specific separation techniques
  
• Gels for specific separation techniques that are made “in-house” or purchased
  
• Buffers for specific separation techniques that are made “in-house” or purchased
  
• Familiarity with suppliers of products and services for protein separation
  
• Estimated percentage of total annual budget for products/services dedicated to protein separation
  
• Current and future use of an integrated proteomics system that includes instrumentation specifically for protein separation

Section 4. Presentation of Survey Data

• Over 40 pages of detailed tables, full-color analytical charts and graphs highlighting the responses and findings for each of the 26 questions in the survey instrument.

Section 5. Cross Tabulations of Survey Data

• Over 55 questions or answer choices tabulated against Market Segment, Geographic Region, Area of Research or other relevant questions.

Section 6. Appendix

• Related Reports
  
• About BioInformatics
  
• Our Valued Clients

Abstract

2-D gel electrophoresis allows protein scientists to separate either a whole cell or tissue protein extracts and then identify these proteins through peptide mass fingerprinting or via direct sequencing utilizing mass spectrometry (MS). Although 2DGE is the most widely used tool for separating proteins in expression proteomics, it is not without its limitations. Challenges faced when utilizing this technology are comigration of proteins, systematic exclusion of highly hydrophobic molecules, and problems with detecting very acidic, very basic, very small, very large, or low abundance proteins.

To meet the demands of technology, companies are developing new innovations that appear to be inexpensive and reliable, generate high-resolution protein separation and yield good visual detection of subtle differences. Today gels are available as homogeneous, pre-cast products, which are combined with automated equipment and imaging analysis software for improved quantitative results and reduced workload. One example of a more advanced proteomic system is a 2-D gel electrophoresis system with common features such as an automated stainer, a 2-D analyzer, an automated gel exciser, an automated digester, and a robot for preMS sample preparation. Finally, as emerging technologies such as microcapillary electrophoresis, capillary electrochromatography and ultra-HPLC begin to replace 2-D gel electrophoresis, companies will need customer feedback in order to develop and maintain a competitive advantage in their product design and production.

The final report is based on the opinions of a worldwide panel of research scientists. It provides you with the answers needed to better serve the protein science market and is available at a fraction of what it would cost to conduct a custom study similar in size and scope.

Protein Separation is the third installment in our series - which details all aspects of proteomics - and specifically focuses on the well-established method of 2-D gel electrophoresis. Protein chemists widely use first-dimensional separation by isoelectric focusing and second-dimensional separation by molecular weight despite existing disadvantages or limitations. As interest in alternatives grow, companies involved in supporting protein research must understand life scientists’ successes and frustrations with “state-of-the-art” technologies and how products must evolve to better meet the needs of this dynamic market.

Report Highlights

More than 500 researchers engaged in protein separation participated in this survey between September 4 and November 23, 2001. The report details findings for each and every question in the survey. Below is a glimpse of some of the findings derived from different questions:

• The most preferred separation technique is one-dimensional SDS-polyacrylamide gel electrophoresis (PAGE) under reducing conditions.
• Most respondents who use multiple separation techniques employ either single or multiple, tandem liquid chromatography approaches with or without subsequent mass spectrometry.
• Over the next twelve months, usage of two cutting-edge approaches to separation technology - capillary gel electrophoresis and microfluidics “lab-on-a-chip” - in conjunction with mass spectrometry will significantly increase.
• Respondents primarily use one-dimensional isoelectric focusing or one-dimensional SDS-PAGE for protein analysis/characterization experiments.
• The protein scientists surveyed prefer to use two-dimensional gel electrophoresis for protein characterization and identification experiments but not for purifying their protein(s).
• Over three-quarters of the scientists do not plan to use an integrated proteomics system that includes instrumentation specifically for protein separation.

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