While traditional multiplexed immunoassays have advanced scientific understanding of intracellular signaling, a number of challenges remain. Chief among these is a lack of sufficient targets for pathway analysis; the delivery of qualitative or relative quantitative results, not absolute quantitative results; and the need for separate wells to analyze each phosphorylated site on a specific analyte, as well as total protein.
A new quantitative approach to multiplex immunoassays simultaneously measures both total protein abundance and site-specific phosphorylation at the peptide level with picomolar sensitivity. This technology is designed to explore signaling pathways affected by epidermal growth factor receptor inhibition.
The ErbB or epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases consists of four members: EGFR/ErbB1/HER1, ErbB2/Neu/HER2, ErbB3/ HER3, and ErbB4/HER4. The ErbB receptors play crucial roles in propagating signals regulating cell proliferation, differentiation, motility, and apoptosis, contributing to pathological processes such as cancer. The ability to analyze the phosphorylation status of ErbB family members―as well as the phosphorylation status of receptor-related intracellular signal transduction proteins―is necessary for a thorough understanding of this signaling pathway.
To examine the phosphorylation status of the ErbB family and receptor-related intracellular proteins, several assay platforms are available. The Western blot has been the gold standard for studying pathway signaling. Other platforms include ELISA, reverse-phase arrays, high content analysis, and mass spectroscopy. Although some of these platforms yield absolute quantitative data, the assay is either limited to measuring one analyte at a time or there is a high level of difficulty and expense.
In recent years, bead-based assays using the Luminex xMAP technology (Luminex Corp.) have enabled researchers to study phosphorylation levels of up to 12 proteins simultaneously. The xMAP platform enables researchers to differentiate dozens of analytes per sample, reducing the time, labor, and costs associated with methods such as ELISA and Western blotting, which are only semi-quantitative and offer limited throughput.
The xMAP platform consists of fluorescently dye-labeled magnetic microspheres, a flow cytometry- or CCD camera-based instrument, and software for data acquisition and analysis. With the xMAP platform, sandwich assays are performed on a bead. Each bead contains two red dyes; 10 different concentrations of the dye yield 100 different possible combinations. Since the beads are in solution rather than fixed to a plate, up to 50 different beads with different capture antibodies can be used with one sample yielding results for up to 50 different proteins. When the beads pass through the reader or chamber, the ratio of the two dyes indicates the bead number.
Streptavidin-phycoerythrin, which fluoresces green, is used as the common detection reagent binding to biotinylated antibodies. The detection system reads red and green, thus providing identification of the analyte and quantitation of the amount bound to the bead.
However, there is a lack of phosphorylation site-specific antibodies; multiple phosphorylation sites on the same protein cannot be interrogated simultaneously; and results obtained are relatively quantitative.
Milliplex MAP EpiQuant technology enables measurement of phosphorylation levels at multiple sites, as well as total protein, simultaneously with absolute quantitation. Based on the Luminex xMAP platform, EpiQuant, from EMD Millipore, uses antibodies against defined protein sequences to capture peptides resulting from protein linearization and digestion in a single cell lysate sample. The fragments generated are similar to those generated using mass spectroscopy. These peptides are then used to generate standard curves, which results in quantitative measurements with picomolar sensitivity.
Using this technology, EMD Millipore constructed a 22-plex immunoassay for the analysis of ErbB signaling pathway constituents. The Milliplex MAP EpiQuant EGFR pathway panel allows the analysis and quantitation of tyrosine phosphorylation sites on ErbB2 and ErbB3 and multiple sites on ErbB1, as well as tyrosine phosphorylation sites for 16 other receptor related proteins, including ERK1/2 and multiple Gab2 and FAK sites.
In addition to containing 20 phosphorylated targets, the assay also quantified total EGFR and a loading control (TAFII68) simultaneously in a single assay well. An internal study demonstrated the use of this assay in the analysis of EGF-treated A431 skin carcinoma cells. Dose- and time-dependent responses were observed across a majority of the analytes in the panel.
Inhibitor studies were performed to provide an example of the panel’s use in cell signaling and cancer research. A431 human epithelial carcinoma cells were cultured according to ATCC guidelines in recommended media. Cells were grown to approximately 85% confluence, then serum starved for 4 hours prior to treatment with stimuli or compounds. Cells were lysed and samples collected according to Milliplex MAP EpiQuant sample preparation kit instructions. The multiplex assay was performed in a 96-well plate.
The procedure is as follows:
1. Rinse the plate with 100 μL assay buffer.
2. Add 25 μL standards/samples, 25 μL beads to each well.
3. Incubate 2 hours at room temperature.
4. Wash the beads two times with assay buffer.
5. Add biotinylated detection antibody cocktail, incubate 1 hour at room temperature.
6. Remove detection cocktail and add 25 μL streptavidin-phycoerythrin (SA-PE), incubate for 30 minutes at room temperature.
7. Remove SA-PE and resuspend beads in 150 μL assay buffer.
8. Read assay plate using Luminex system.
The Milliplex MAP EpiQuant EGFR pathway magnetic bead panel enabled the detection of phosphorylation events for all panel analytes in dose response and time-course experiments. The TAFII68 loading control values obtained were used to normalize the amount of lysate for each well.
Figure 2 illustrates the results of the dose-response experiments for several targets. Of interest is the observation that concentration of EGF needed for maximal activation of the two EGFR phosphorylation sites appears dissimilar.
Figure 3 shows the results of the time course experiments. The perpetuation of maximal signal is observed for many targets (ERK1/2 and EGFR, for example) across the whole of the time course, while for other targets (FRS2 and PKCμ, for example) maximal signal levels are observed at the 5-minute time point with a return to basal levels by 20 to 30 minutes. As would be expected due to receptor internalization, it appears as though total EGFR levels drop after stimulation, with a rebound to less than resting levels at the one hour time point.
Figure 4 demonstrates the effect of gefitinib treatment on phosphorylation levels for several targets. Gefitinib is an EGFR-selective inhibitor and chemotherapeutic agent indicated for the treatment of non-small cell lung carcinoma. A dose-dependent response is observed in phosphoprotein levels, while total EGFR concentrations remain relatively constant.
The complexity and number of protein targets involved in signaling events, as well as cellular responses, require tools that enable multiplex analysis of samples. This approach enables analysis of a greater number of intracellular analytes per well and simultaneous measurement of multiple phosphorylation sites with picomolar sensitivities, absolute quantitation of multiple phosphorylation sites on the same protein, and absolute quantitation of both total and phosphoproteins.
About the Author
Rick Wiese has worked on both bead-based and planar protein array platforms for over 10 years, and has more than 15 years experience in cellular signaling and toxicology, primarily in the areas of xenobiotic metabolism and carcinogenesis.