In preclinical proteomics, speed and accuracy are paramount. Increasingly, scientists must capture more data in less time while consuming minimal sample. Given the throughputs required for efficient drug discovery projects, biomarker analysis, and RNAi knockdown confirmation—to name only a few applications—the need for rapid protein quantitation has reached a critical point.
The Luminex® (www.luminexcorp.com) xMAP® platform is often used for such analyses. Luminex xMAP assays are essentially immunosandwich ELISAs on a microparticle bead detected with the aid of a modified flow cytometer. Unlike traditional ELISAs, though, dozens of different protein targets may be quantified simultaneously from a single sample well.
This is possible because each bead type (conjugated to a specific capture antibody) has a uniquely identifiable dye signature. The xMAP instrument detects signal from biotinylated secondary antibodies used in combination with streptavidin-phycoerythrin; it also cross-correlates capture-bead signal with phycoerythrin signal. Fitting the signals from experimental samples to standard curves enables quantification.
Rapidity is a distinct advantage of the xMAP assay format. In comparison to 2-D platforms such as microarrays, which are limited by solid-phase kinetics, xMAP assays approach solution-phase kinetics. The sensitivity is equal to or exceeding that of traditional ELISAs. The ability to multiplex is an advantage, saving precious sample and enabling analyses that would be either too laborious or weakened by statistical noise if the samples were instead processed in parallel.
Unlike platforms such as arrays that rely on image analysis, storage space required for xMAP data is minimal—a considerable logistical advantage for large screening projects. Novagen® WideScreen™ Assays from EMD Chemicals (www.novagen.com/widescreen) for the Luminex xMAP platform enable quantitation of key signaling proteins. In this article, we describe two panels: the WideScreen Receptor Tyrosine Kinase (RTK) Assays and the WideScreen EpiTag™ Extracellular Signal-Regulated Kinase (ERK) Assays.
Receptor Tyrosine Kinases
The WideScreen RTK panel includes comprehensive RTK target coverage for the Luminex xMAP platform. RTKs are transmembrane cell-surface kinases activated upon binding extracellular ligands. They are of great interest due to their control of critical developmental and disease processes.
The Novagen WideScreen RTK Assay allows single or multiplex analysis of key RTKs including EGFR, IGF-1R, HGFR, PDGFRb, HER-2, VEGFR2, and Tie-2. The oncological roles of several of these receptors have been intensely scrutinized.
For example, EGFR overexpression or mutation has been correlated with numerous solid organ tumors including colorectal, pancreatic, lung, and non-small-cell lung cancers and glioblastoma. HER-2 overexpression (occurring in 25% of breast cancer patients) is associated with poor prognosis and disease recurrence, while VEGFR2 regulates angiogenesis pathways.
WideScreen RTK Assays consist of a series of phosphotyrosine (pTyr)-specific RTK assays and companion Total (phosphorylated plus unphosphorylated) assays. pTyr assays utilize specific capture antibodies and a broad-spectrum, pTyr-detection antibody. Total RTK assays, which include standards, allow relative comparison of pTyr and Total RTK in the sample for each receptor when Total and pTyr assays are conducted in parallel.
WideScreen RTK Assay Kits include an optimized buffer for detergent-mediated lysis, eliminating the need to alter lysate preparation for each antibody/analyte combination. Complete Kits include all seven premixed, antibody-conjugated capture beads, detection antibodies, total RTK standards, extraction reagents, and wash buffers. Single Bead Kits are available for researchers wishing to define their own multiplex assay combinations.
WideScreen RTK assay applications include protein-expression profiling, RNAi knock-down experiment confirmation, biomarker quantitation, inhibitor profiling, pathway analysis, and compound screening. Figure 1A shows an example of protein-expression profiling conducted with the WideScreen RTK Total Assay Complete Kit.
In this experiment, HUVEC and HT-29 cell-line extracts were analyzed for protein-expression levels of all seven RTKs in the panel. The results indicate marked differences in RTK protein expression between the two lines. HUVEC cells showed a high expression of Tie-2, as has been previously reported. HT-29 human colon cancer cells displayed a characteristically high level of HER-2 protein expression.
Figure 1B demonstrates the use of the WideScreen pTyr Assay to assess the response of serum-starved HT-29 cells to increasing concentrations of PD 168393, known to be highly specific for EGFR and HER-2.
ERK/MAPK Signaling Pathway
The ERK/MAPK pathway is an integral regulator of the cell cycle and apoptosis; it has recently been a subject of target-based therapy development. Some of the earliest ERK/MAPK inhibitory agents have received FDA approval for chemotherapeutic use, while numerous others are the subject of ongoing clinical trials.
WideScreen EpiTag ERK Assays utilize technology licensed from Epitome Biosystems (www.epitomebiosystems.com), which detects protein and phosphoprotein fragments rather than intact proteins. In this method, cell or tissue lysates are proteolytically digested, and antibody pairs to unique amino acid sequences (EpiTag Sequences) permit specific and sensitive target detection. Thus, even closely related analytes such as ERK1 and ERK2 can be discerned with precision. The included synthetic peptide or phosphopeptide standards permit absolute quantification of total or phosphorylated forms of target proteins. This approach also allows phospho and total assays to be multiplexed together in the same assay well.
Figure 2 shows the utility of the WideScreen EpiTag ERK Assays for pathway analysis during inhibitor profiling. Serum-starved A-431 cells were either unstimulated or stimulated with recombinant human epidermal growth factor (rh-EGF) in the presence or absence of the MEK1/2 activity inhibitor SL327, a structural analog of U0126. Samples were analyzed using the WideScreen EpiTag ERK Pathway Panel I Complete Kit.
Whereas MEK1/2, ERK1, and ERK2 showed increased phosphorylation upon rh-EGF stimulation, the level of phospho-B-Raf was constitutive, as has been described in the literature. Stimulation of A-431 cells with rh-EGF increased phosphorylation of MEK1/2 over ninefold.
Addition of 4 mM SL327 reduced phosphorylation of MEK1/2 and downstream components ERK1 and ERK2. Inhibitor-titration experiments using U-87 cells (Figure 3) also confirmed that SL327 selectively targets the activity of MEK1/2 and thereby substantially influences ERK1 and ERK2 phosphorylation levels, with observed IC50 values corresponding to that reported in the literature. As expected, the phosphorylation of upstream component B-Raf was not impacted.
WideScreen RTK Assays and WideScreen EpiTag ERK Assays for the Luminex xMAP platform are reliable and robust tools for quantitation of key cell-signaling components. The documentation supporting these kits includes performance characteristics for each antibody like analyte specificity, phosphospecificity, intra- and inter-assay specificity, and lot-specific quality-control verification.