September 15, 2012 (Vol. 32, No. 16)

Novel Technology Takes Aim at Improving Phosphorylation Level Measurement

Errors in cell-signaling pathways play a central role in many diseases including cancer, diabetes, and autoimmune disorders. The ability to study these signaling pathways and accurately measure protein and protein phosphorylation levels is increasingly important in drug discovery and the development of disease models.

Traditional multiplexed immunoassays such as those conducted on the Luminex® xMAP® platform have advanced our understanding of intracellular communication by enabling the ability to measure multiple total or phosphorylated sites along a signaling pathway in a single sample, and simultaneously measure sites in multiple pathways. However, multiplexing still presents some challenges as there is a lack of phosphospecific antibodies that work well on the Luminex xMAP platform due to:

  • The requirement for native protein confirmation instead of recognition of denatured proteins on Western blots
  • Complex buffer interactions with phosphoproteins
  • Matrix and inter-antibody effects when multiplexed

Resulting difficulties include a lack of sufficient targets for pathway analysis, qualitative or relative quantitative results, and the need for separate wells to analyze each phosphorylated site on a specific analyte as well as total protein.

To overcome these challenges, the MILLIPLEX® MAP EpiQuant™ receptor signaling portfolio (EMD Millipore) enables researchers to measure phosphorylation levels of multiple sites, as well as total protein, simultaneously with absolute quantitation.

Based on the Luminex xMAP platform, the technology 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 same peptides are then used as standards to generate curves with picomolar sensitivity.

This multiplexing technology provides faster answers to cell signaling questions compared to traditional Western blots, mass spectrometry analyses, and radioactive phosphorylation assays requiring large amounts of resources, time, and sample.

Figure 1. Analysis of A431 human epithelial carcinoma cells treated with EGF, H2O2 or vehicle.

EGFR Pathway Analysis

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.

The MILLIPLEX MAP EpiQuant EGFR pathway panel profiles 29 ErbB signaling pathway constituents. The assay enables 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 is also able to quantify total EGFR and a loading control (TAFII68) simultaneously in a single assay well.

Figure 1 shows results of an analysis of A431 human epithelial carcinoma cells treated with EGF, H2O2, or vehicle. Following lysis and digestion, EpiQuant™ beads (a 22-plex) were added to samples containing the target proteins shown. Samples were analyzed on a Luminex 200™ reader. Data shown are the mean pM values from four observations. Increased phosphorylation was noted for EGFR, Her2, IL-15R, Shc, and Tec compared to vehicle-treated cells.

The EGFR pathway panel enables the detection of phosphorylation events for all panel analytes in dose response and time course experiments. Figure 2 illustrates the results of a dose response experiment 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 2. Simultaneous detection of phosphorylated EGFR pathway proteins as well as total EGFR.

Inflammation Pathway Analysis

Signaling events initiated by extracellular receptor activation lead to the expression and release of cytokines/chemokines. These events are central to cellular and tissue homeostasis, and developmental and functional response. The complexity and number of signaling proteins involved in cellular responses require multiplex analysis of samples.

A549 lung carcinoma cells were cultured, and then stimulated with either EGF or TNFα. For the cytokine/chemokine analysis, tissue culture media were collected at various time points over a 24-hour period, as well as unstimulated controls. In addition, cell lysates were collected at time points over a one-hour period. Prepared samples were assayed according to the MILLIPLEX MAP Human Cytokine/Chemokine-42-plex protocol and the MILLIPLEX MAP EpiQuant Phosphotyrosine Cell Signaling Panel 1 protocol, respectively. A total of 42 soluble analytes and 105 intracellular phosphotyrosine proteins were analyzed (Figure 3).

Analysis of TNFα-stimulated A549 supernatant revealed an increased expression in 21 of the 42 screened cytokine/chemokine analytes. Of the 105 phosphotyrosine cell signaling targets analyzed, a response was observed in 38 target sites for TNFα− or EGF-treated cells. The magnitude of cytokine/chemokine and phosphotyrosine response varied significantly between targets. For several phosphorylation sites, a decrease in phosphorylation was observed in response to TNFα treatment.

The complexity and number of protein targets involved in signaling events, as well as cellular responses, require tools that enable multiplex analysis of samples. MILLIPLEX MAP EpiQuant technology enables the analysis of a greater number of intracellular analytes per well compared to even traditional Luminex intracellular assays. The magnetic bead format of these assays provides additional convenience with walk-away washing options and increased consistency, resulting in lower coefficients of variation.

Figure 3. Site-specific phosphotyrosine concentrations over time for several responsive phosphoproteins.

Rick Wiese, Ph.D. ([email protected]), is R&D manager at EMD Millipore.

Previous articleBayer HealthCare to Buy Teva Animal Health Business
Next articleFeatured Video: Mastering the DNA Transfection Workflow