Shirshendu K. Deb

Detection and relative quantification of proteins by surface enhanced Raman using isotopic labels.

Accurate quantification of protein content and composition has been achieved using isotope-edited surface enhanced resonance Raman spectroscopy. Synthesis of isotopomeric Rhodamine dye-linked bioconjugation reagents enabled direct labeling of surface lysines on a variety of proteins. When separated in polyacrylamide gels and stained with silver nanoparticles. The spectral signatures reflect the expected statistical distribution of isotopomeric labels on the labeled proteins in the gel matrix format without interference from protein features.

Accurate Concentration Measurements Using Surface-Enhanced Raman and Deuterium Exchanged Dye Pairs

Quantitative applications of surface-enhanced resonance Raman scattering (SERRS) are often limited by the reproducibility of SERRS intensities, given the difficulty of controlling analyte–substrate interactions and the associated local field enhancement. As demonstrated here, SERRS from dye molecules even within the same structural class that compete with similar substrates display distinct spectral intensities that are not proportional to analyte concentrations, which limits their use as internal standardization probes and/or for multiplex analysis. Recently, we demonstrated that isotopic variants of rhodamine 6G (R6G), namely R6G-d0 and R6G-d4, can be used for internal standards in SERRS experiments with a linear optical response from picomolar to micromolar concentrations (of total analytes). Here we extend these results by describing a straightforward method for obtaining isotopomeric pairs of other Raman active dyes by hydrogen–deuterium exchange conditions for substitution at electron rich aromatic heterocycles. Most of the known SERRS active probes can be converted into the corresponding isotopomeric molecule by this exchange method, which significantly expands the scope of the isotopic edited internal standard (IEIS) approach. The relative quantification using IEIS enables accurate, reproducible (residual standard deviation ±2.2%) concentration measurements over a range of 200 pM to 2 μM. These studies enable easy access to a variety of isotopically substituted Raman active dyes and establish the generality of the methodology for quantitative SERRS measurements. For the first time, three rhodamine 6G isotopomers have been created and show distinct Raman spectra, demonstrating the principle of the approach for application as a multiplex technique in biomolecular detection/quantification.

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

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.