2D DIGE Saturation Labeling for Minute Sample Amounts - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 89

Chapter 7
2D DIGE Saturation Labeling for Minute Sample Amounts
Georg J. Arnold and Thomas Fröhlich
Abstract
The 2D DIGE technique, based on fl uorophores covalently linked to amino acid side chain residues and
the concept of an internal standard, has signifi cantly improved reproducibility, sensitivity, and the dynamic
range of protein quantifi cation. In saturation DIGE, sulfhydryl groups of cysteines are labeled with cyanine
dyes to completion, providing a so far unraveled sensitivity for protein detection and quantifi cation in 2D
gel-based proteomic experiments. Only a few micrograms of protein per 2D gel facilitate the analysis of
about 2,000 analytes from complex mammalian cell or tissue samples. As a consequence, 2D saturation
DIGE is the method of choice when only minute sample amounts are available for quantitative proteome
analysis at the level of proteins rather than peptides.
Since very low amounts of samples have to be handled in a reproducible manner, saturation DIGE-
based proteomic experiments are technically demanding. Moreover, successful saturation DIGE approaches
require a strict adherence to adequate reaction conditions at each step. This chapter is dedicated to col-
leagues already experienced in 2D PAGE protein separation and intends to support the establishment of
this ultrasensitive technique in proteomic workgroups. We provide basic guidelines for the experimental
design and discuss crucial aspects concerning labeling chemistry, sample preparation, and pitfalls caused by
labeling artifacts. A detailed step-by-step protocol comprises all aspects from initial sample preparation to
image analysis and statistical evaluation. Furthermore, we describe the generation of preparative saturation
DIGE gels necessary for mass spectrometry-based spot identifi cation.
Key words: Quantifi cation, Protein, Proteomics, 2D DIGE, Saturation labeling, CyDyes
1. Introduction
Current proteomic workfl ows use chromatographic or electrophoretic
methods for separation of proteins or peptides from complex mix-
tures before identifi cation in a mass spectrometer. Two-dimensional
(2D) gel electrophoresis is a well-established technique, facilitating
the separation of around 2,000 protein analytes in a standard gel.
A special benefi t of this method is the convenient detection and
quantifi cation of protein isoforms or modifi cations which differ in
the molecular mass or the isoelectric point. Quantifi cation of