Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology

¹Department of Molecular Biotechnology, University of Washington, Seattle, WA 98195-7730, USA.

|August 2, 2000

Summary

Two-dimensional gel electrophoresis coupled with mass spectrometry (2DE-MS) struggles to identify low-abundance proteins. This proteomic technique

Area of Science:

Background:

Proteome analysis commonly uses two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS).
Despite its power, the 2DE-MS technique faces limitations in comprehensive proteome characterization.
Questions persist regarding the sensitivity of 2DE-MS for detecting all proteomic elements.

Purpose of the Study:

To evaluate the capability of the 2DE-MS approach for identifying low-abundance proteins.
To investigate the limitations of 2DE-MS in comprehensive proteome analysis.
To determine if protein expression levels impact the detection of low-abundance proteins.

Main Methods:

Separation and visualization of proteins using silver-stained 2DE gels within a narrow pH range (4.9-5.7).
Analysis of approximately 1,500 visualized protein features from 0.5 mg of total soluble yeast protein.
Protein identification of 50 selected spots using mass spectrometry (MS).
Fractionation and analysis of protein amounts exceeding 2DE capacity.

Main Results:

Proteins from genes with low codon bias values (<0.1), indicative of lower abundance, were not identified by standard 2DE-MS.
These low-abundance proteins represent approximately 50% of all yeast genes.
Low-abundance proteins were detected when sample amounts exceeded the capacity of 2DE, requiring fractionation.

Conclusions:

The wide dynamic range of protein expression levels inherently limits the 2DE-MS technique's ability to analyze medium to low-abundance proteins.
The comprehensive potential of 2DE-MS for complete proteome analysis is constrained by its detection limits for less abundant proteins.

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