title: 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.
authors: Respiratory Medicine Unit, Department of Medicine, Center for Molecular Medicine, Karolinska Institutet , 171 76 Stockholm, Sweden.
authors: AnnSofi
authors: A
authors: Sandberg
authors: PicoQuant GmbH, Rudower Chaussee 29, 124 89 Berlin, Germany.
authors: Volker
authors: V
authors: Buschmann
authors: PicoQuant GmbH, Rudower Chaussee 29, 124 89 Berlin, Germany.
authors: Peter
authors: P
authors: Kapusta
authors: PicoQuant GmbH, Rudower Chaussee 29, 124 89 Berlin, Germany.
authors: Rainer
authors: R
authors: Erdmann
authors: Respiratory Medicine Unit, Department of Medicine, Center for Molecular Medicine, Karolinska Institutet , 171 76 Stockholm, Sweden.|Hikari Bio AB, Franzéngatan 39, 112 16 Stockholm, Sweden.
authors: Åsa M
authors: ÅM
authors: Wheelock
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D057230:Limit of Detection; D040901:Proteomics