Quantification of isotope encoded proteins in 2-D gels using surface enhanced resonance Raman.

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.

• Authors: Giselle M Knudsen, Brandon M Davis, Shirshendu K Deb, Yvette Loethen, Ravindra Gudihal, Pradeep Perera, Dor Ben-Amotz, V Jo Davisson
• Journal: Bioconjugate chemistry (Bioconjug Chem) Vol. 19 Issue 11 Pg. 2212-20 (Nov 19 2008) ISSN: 1520-4812 [Electronic] United States
• PMID: 18925772 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, U.S. Gov't, Non-P.H.S.)

Chemical References

• Cell Extracts
• Esters
• Protein Isoforms
• Proteins
• Rhodamines
• rhodamine 6G
• Silver
• Carbon-Nitrogen Ligases
• GMP synthase (glutamine-hydrolyzing)

Topics

• Animals
• Carbon-Nitrogen Ligases (analysis, chemistry, metabolism)
• Cell Extracts (chemistry)
• Cell Line, Tumor
• Electrophoresis, Gel, Two-Dimensional
• Esters (chemistry)
• Fluorescence
• Humans
• Isotope Labeling
• Protein Isoforms (analysis, chemistry)
• Proteins (analysis, chemistry)
• Reference Standards
• Reproducibility of Results
• Rhodamines (chemistry, metabolism)
• Sensitivity and Specificity
• Silver (chemistry)
• Spectrum Analysis, Raman