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High dynamic range proteome imaging with the structured illumination gel imager
Phu T Van1, Victor Bass, Dan Shiwarski
1Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA.
Electrophoresis
|June 18, 2014
Summary
Detecting proteins across vast concentration differences in biological samples is challenging. A new structured illumination (SI) gel imager overcomes this by enabling unbiased, whole-proteome analysis with a 1,000,000-fold dynamic range.
Area of Science:
- Proteomics
- Biotechnology
- Analytical Chemistry
Background:
- Proteomics faces challenges in detecting proteins across wide concentration ranges in complex biological samples like whole-cell extracts.
- Current unbiased, whole-proteome analysis methods are limited by the dynamic range of available detectors, preventing the detection of the full spectrum of cellular proteins.
Purpose of the Study:
- To introduce a novel technology, the structured illumination (SI) gel imager, designed to overcome the dynamic range limitations in protein detection.
- To enable comprehensive and unbiased proteome-wide surveys by accurately detecting proteins across a significantly expanded concentration range.
Main Methods:
- Development of a structured illumination (SI) gel imager utilizing computer-generated masks to control illumination intensity.
- Application of SI technology to attenuate illumination for highly abundant proteins, enabling longer exposures for low-abundance proteins and preventing detector saturation.
- Assembly of multiple, progressively masked gel images into a single, high dynamic range image for comprehensive analysis.
Main Results:
- Demonstrated the capability of the SI imager to detect fluorescently labeled proteins over an unprecedented 1,000,000-fold concentration range.
- Successfully overcame detector saturation issues inherent in analyzing samples with extreme protein concentration variations.
- Validated the SI imager as a powerful tool for detailed proteome-wide investigations.
Conclusions:
- The structured illumination gel imager significantly advances the field of proteomics by providing a much-needed high dynamic range detection capability.
- This technology facilitates more comprehensive and unbiased analysis of complex proteomes, revealing previously undetectable low-abundance proteins.
- The SI imager represents a critical step towards achieving a complete understanding of cellular protein expression.
