Use of Time-Resolved Fluorescence To Improve Sensitivity and Dynamic Range of Gel-Based Proteomics

Abstract

Limitations in the sensitivity and dynamic range of two-dimensional gel electrophoresis (2-DE) are currently hampering its utility in global proteomics and biomarker discovery applications. In the current study, we present proof-of-concept analyses showing that introducing time-resolved fluorescence in the image acquisition step of in-gel protein quantification provides a sensitive and accurate method for subtracting confounding background fluorescence at the photon level. In-gel protein detection using the minimal difference gel electrophoresis workflow showed improvements in lowest limit of quantification in terms of CyDye molecules per pixel of 330-fold in the blue-green region (Cy2) and 8000-fold in the red region (Cy5) over conventional state-of-the-art image acquisition instrumentation, here represented by the Typhoon 9400 instrument. These improvements make possible the detection of low-abundance proteins present at sub-attomolar levels, thereby representing a quantum leap for the use of gel-based proteomics in biomarker discovery. These improvements were achieved using significantly lower laser powers and overall excitation times, thereby drastically decreasing photobleaching during repeated scanning. The single-fluorochrome detection limits achieved by the cumulative time-resolved emission two-dimensional electrophoresis (CuTEDGE) technology facilitates in-depth proteomics characterization of very scarce samples, for example, primary human tissue materials collected in clinical studies. The unique information provided by high-sensitivity 2-DE, including positional shifts due to post-translational modifications, may increase the chance to detect biomarker signatures of relevance for identification of disease subphenotypes.

Coomassie staining provides routine (sub)femtomole in‐gel detection of intact proteoforms: Expanding opportunities for genuine Top‐down Proteomics

“…Accurate assessments of the concentration and purity of protein standards are necessary for meaningful determinations of stain sensitivity and selectivity. Apparent impurity of commercial protein preparations has been seen to varying degrees , and noted in other investigations in which protein concentration and purity were assessed . Yet it remains a problem in the field that efforts to examine the content/purity of commercial protein standards (and detailing methods to do so) are rarely reported if carried out at all.…”

“…However, such instrumentation, which generally utilise lamps and LEDs rather than lasers, may be expected to be less sensitive (depending on the fluorophore used) due to broader wavelength excitation and emission hardware. Due focus should continue to be placed on improving imaging and analysis approaches in order to maximise outcomes of proteomic investigations, as recently demonstrated by a number of groups . It is clear that the capacity for proteome coverage and detection using 2DE and cCBB staining (and thus likely many other stains) has in general been grossly underestimated .…”