Use of Time-Resolved Fluorescence To Improve Sensitivity and Dynamic Range of Gel-Based Proteomics
| العنوان: | Use of Time-Resolved Fluorescence To Improve Sensitivity and Dynamic Range of Gel-Based Proteomics |
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| المؤلفون: | Peter Kapusta, Rainer Erdmann, Åsa M. Wheelock, AnnSofi Sandberg, Volker Buschmann |
| المصدر: | Analytical Chemistry. 88:3067-3074 |
| بيانات النشر: | American Chemical Society (ACS), 2016. |
| سنة النشر: | 2016 |
| مصطلحات موضوعية: | Proteomics, 0301 basic medicine, Gel electrophoresis, Detection limit, Pixel, Dynamic range, Chemistry, Difference gel electrophoresis, Quantitative proteomics, Analytical chemistry, 02 engineering and technology, 021001 nanoscience & nanotechnology, Fluorescence, Analytical Chemistry, 03 medical and health sciences, 030104 developmental biology, Limit of Detection, Electrophoresis, Polyacrylamide Gel, Biomarker discovery, 0210 nano-technology, Biological system |
| الوصف: | 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. |
| تدمد: | 1520-6882 0003-2700 |
| DOI: | 10.1021/acs.analchem.5b03805 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::a82f4e6a540cc08888189328d817779b https://doi.org/10.1021/acs.analchem.5b03805 |
| رقم الانضمام: | edsair.doi.dedup.....a82f4e6a540cc08888189328d817779b |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 15206882 00032700 |
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| DOI: | 10.1021/acs.analchem.5b03805 |