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Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification
Published on: November 15, 2017
Prefractionation techniques in proteome analysis: the mining tools of the third millennium
Pier Giorgio Righetti1, Annalisa Castagna, Paolo Antonioli
1University of Verona, Department of Industrial and Agricultural Biotechnolgies, Verona, Italy. righetti@sci.univr.it
Electrophoresis
|January 20, 2005
Summary
This review explores prefractionation techniques for proteomics, enhancing protein identification by mass spectrometry (MS) and 2-DE. Novel chromatographic and electrophoretic methods, along with sample treatment strategies, improve detection of low-abundance species.
Area of Science:
- Proteomics and analytical biochemistry
- Biophysical and biochemical separation sciences
Background:
- Proteomic investigations often face challenges in identifying low-abundance proteins due to sample complexity.
- Existing prefractionation methods are crucial for enhancing the sensitivity and scope of protein analysis.
- Mass spectrometry (MS) and two-dimensional electrophoresis (2-DE) are key techniques requiring effective sample preparation.
Purpose of the Study:
- To review and discuss various prefractionation protocols for proteomic analysis.
- To highlight novel chromatographic and electrophoretic techniques for sample simplification and concentration.
- To present strategies for detecting low-abundance proteins in complex biological samples.
Main Methods:
- Differential centrifugation and various chromatographic approaches (affinity, ion-exchange, reversed-phase).
- Novel multistaged fractionation columns utilizing immobilized chemistries.
- Electrophoretic methods in free solution, including isoelectric focusing, and multicompartment electrolyzers.
- Sample treatment using combinatorial ligand libraries to reduce protein concentration differences.
Main Results:
- Chromatographic methods revealed hundreds of previously undetected protein species.
- Multistaged fractionation columns simplify samples and concentrate proteins, increasing MS/2-DE visibility.
- Electrophoretic techniques, particularly isoelectric focusing, offer effective separation in free solution.
- Ligand-based sample treatments significantly reduce concentration disparities, enhancing low-abundance species detection.
Conclusions:
- Prefractionation is essential for comprehensive proteomic analysis, enabling deeper insights into the 'unseen proteome'.
- Advanced chromatographic and electrophoretic techniques offer powerful tools for sample complexity reduction and protein enrichment.
- Strategies for reducing concentration differences are critical for discovering low-abundance biomarkers and proteins.
