High Dynamic Range Imaging Of Polyacrylamide Gels For Proteomic Exploration

The range of protein concentrations in living cells is vast, approximately 100,000-fold. This poses significant problems to comparative proteomic studies where the goal is to detect protein differences between cells across different conditions. We have developed an imaging device, referred to as Structured Illumination Gel Imager (SIGI), that is designed to detect proteins over a million-fold concentration range in an electrophoretic gel. Scientific-grade, Peltier-cooled, 16-bit CCD cameras can distinguish 65,535 gray levels, or ~10,000-fold protein concentration range. Beyond this range gel images saturate, where brightest pixels have a value of 65,535 regardless of true brightness, making longer exposures uninformative and severely limiting dynamic range. SIGI extends dynamic range through structured illumination: an LCD projector projects a binary mask where regions of the gel with low-abundance proteins receive full illumination while regions with high-abundance proteins receive no illumination, preventing high-abundance spots from saturating the CCD camera, thus enabling longer exposures. Applied iteratively, structured illumination allows SIGI to capture exposures many times longer than typical single-exposure images taken without masking. In our experiments SIGI detected proteins from 10 picograms to 10 micrograms in the same image of a typical polyacrylamide gel, a dynamic range of 1,000,000 fold.

SIGI’s projected masks also reduce light scatter, making it possible to visualize low-abundance proteins close to high-abundant proteins in 2DE gels. Quantification of 2DE gels using SIGI as compared to conventional fluorescence imaging shows a more than 50% increase in the number of detected protein spots. These results show SIGI’s potential for peering more deeply into the proteome than previously possible.