Quantification of Isotope Encoded Proteins in 2-D Gels Using Surface Enhanced Resonance Raman

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• 作者：Giselle M. Knudsen ; Brandon M. Davis ; Shirshendu K. Deb ; Yvette Loethen ; Ravindra Gudihal ; Pradeep Perera ; Dor Ben-Amotz ; V. Jo Davisson
• 刊名：Bioconjugate Chemistry
• 出版年：2008
• 出版时间：November 19, 2008
• 年：2008
• 卷：19
• 期：11
• 页码：2212-2220
• 全文大小：311K
• 年卷期：v.19,no.11(November 19, 2008)
• ISSN：1520-4812

文摘

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.