Quantification of isotope encoded proteins in 2-D gels using surface enhanced resonance Raman |
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Authors: | Knudsen Giselle M Davis Brandon M Deb Shirshendu K Loethen Yvette Gudihal Ravindra Perera Pradeep Ben-Amotz Dor Davisson V Jo |
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Affiliation: | Department of Medicinal Chemistry, Bindley Bioscience Center at Discovery Park, Purdue University, West Lafayette, Indiana 47907, USA. |
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Abstract: | A strategy for quantification of multiple protein isoforms from a complex sample background is demonstrated, combining isotopomeric rhodamine 6G (R6G) labels and surface-enhanced Raman in polyacrylamide matrix. The procedure involves isotope-encoding by lysine-labeling with (R6G) active ester reagents, isoform separation by 2-DGE, fluorescence quantification using internal standardization to water, and silver nanoparticle deposition followed by surface-enhanced Raman detection. R6G sample encoding and standardization enabled the determination of total protein concentration and the distribution of specific isoforms using the combined detection approach of water-referenced fluorescence spectral imaging and ratiometric quantification. A detection limit of approximately 13.5 picomolar R6G-labeled protein was determined for the surface-enhanced Raman in a gel matrix (15-fold lower than fluorescence). High quantification accuracies for small differences in protein populations at low nanogram abundance were demonstrated for human GMP synthetase (hGMPS) either as purified protein samples in a single-point determination mode (3% relative standard deviation, RSD%) or as HCT116 human cancer cellular lysate in an imaging application (with 16% RSD%). These results represent a prototype for future applications of isotopic surface-enhanced resonance Raman scatter to quantification of protein distributions. |
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