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Exploring EGFR SignalingUsing the MILLIPLEX® map EpiQuant™ EGFR Pathway Magnetic Bead Panel

Rick Wiese, Ph.D., manages the MILLIPLEX® cell signaling research and development group of EMD Millipore in St Charles, Missouri, USA

Dr. Wiese has worked on both bead-based and planar protein array platforms for over 10 years, currently managing the development of the MILLIPLEX® MAPmates™ and MILLIPLEX® EpiQuant™ product lines. He has more than 15 years experience in cellular signaling and toxicology, primarily in the areas of xenobiotic metabolism and carcinogenesis.

IntroductionThe 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 this end, we constructed a 22-plex immunoassay for the analysis of ErbB signaling pathway constituents. The assay enables for 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. Here we demonstrate 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. Additionally, inhibitor studies were performed. These results clearly demonstrate the utility of this assay and provide an example of the panel’s use in cell signaling and cancer research.

Cellsurvival

mTOR

pAKT

CREB

NFkB

JAK

STAT*

Cellsurvival

Cellsurvival

Proteinsynthesis

Proteinsynthesis

PI3 kinase/AKTpathway

JAK/STAT pathwayERK pathway

EGF

Receptor*

elF2B

active

Inactive

Shc*

EGFR Signaling Pathway

ERK*

RAS

pGSK3

Figure 1.The EGFR signaling pathway is represented in this figure. Several MILLIPLEX® map EpiQuant™ EGFR pathway magnetic bead panel analytes are highlighted*.

EGFR Signaling Pathway

MILLIPLEX® mag EpiQuant™ EGFR Pathway Magnetic Bead Panel Analyte List

Csk (Tyr304) ERK1/2 (Tyr204/187) Gab1 (Tyr285/317) HER2 (Tyr1023) PLCγ2 (Tyr1197/1217)

EGFR (Tyr1110/1125) FAK (Tyr397/407) Gab2 (Tyr266) PKCµ (Tyr502) Shc (Tyr349/350)

EGFR (Tyr1069/1092) FAK (Tyr861) Gab2 (Tyr584) Pl3Kp110δ (Tyr485) SPRY2 (Tyr55)

ErbB3 (Tyr1197/99/1262/76/89/1307) FRS2 (Tyr436) Gab2 (Tyr614) PLCγ1 (Tyr1253) STAT3 (Tyr705)

MethodsLuminex® 200™ System. This is a compact unit consisting of an analyzer, a computer, and software.

Microspheres. Microsphere beads were purchased from Luminex Corporation. Each set of beads is distinguished by different ratios of two internal dyes yielding a unique fluorescent signature to each bead set. Capture antibodies were covalently coupled to the carboxylate-modified microsphere beads.

Tissue Culture. A431 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.

Sample Preparation. Cells were lysed and samples collected according to MILLIPLEX® map EpiQuant™ sample preparation kit (Cat. No. MPEQ-SP) instructions.

Immunoassay Protocol. The multiplex assay was performed in a 96-well plate according to product instructions supplied for the MILLIPLEX® map EGFR panel (Cat No. MPEQMAG-110K or MPEQMG-110K-PX22). The detailed 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 2h RT

4. Wash the beads two times with assay buffer

5. Add biotinylated detection Ab cocktail, incubate 1h RT

6. Remove detection cocktail and add 25 μL Streptavidin-Phycoerythrin (SA-PE), incubate 30 min RT

7. Remove SAPE and resuspend beads in 150 μL assay buffer

8. Read assay plate using Luminex instrumentation.

Results and DiscussionThe 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 illustrates 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 stimula-tion, with a rebound to less than resting levels at the one hour time point. Finally, Figure 4 illustrates the effect of gefitinib treatment on phosphorylation levels for several targets. Gefitinib is an EGFR-selective inhibitor and chemotherapeutic agent, marketed under the name of Iressa®, and is indicated for the treatment of NSCLC (non-small cell lung carcinoma). A dose-dependent response is observed in phosphoprotein levels, while total EGFR concentrations remain relatively constant. The obtained results clearly demonstrate the utility and sensitivity of the EpiQuant™ EGFR 22-plex panel.

EpiQuant™ EGFR Panel: EGF Dose Response

[Ana

lyte

] (pM

)

[EGF] (ng/mL)

11 10 100 1000 10000

10

100

1,000

10,000

EGFR (pY1110/1125) ERK1/2 (pY204/187)

EGFR (pY1069/1092) Shc (pY349/350)

ErbB3 (pY1197/1307)

Figure 2. Phosphorylated EGFR pathway proteins, as well as total EGFR, were simultaneously detected in A431 cells treated with 1000, 333, 111, 37,12.3 and 0 ng/mL EGF. Lysates were collected after 5 minutes EGF stimulation. Values are internally normalized utilizing the TAFII68 loading control.

EpiQuant™ EGFR Panel: EGF Time Course

[Ana

lyte

] (pM

)

Time (min)

10 10 20 30 40 50 60 70

1000

100

10

EGFR (pY1110/1125) HER2 (pY1023)

ErbB3 (pY1197/1307) EGFR total

EGFR (pY1069/1092)

[Ana

lyte

] (pM

)

Time (min)

0.10 10 20 30 40 50 60 70

100

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1

FRS2 (pY436) Gab2 (pY614)

Gab2 (pY584) Gab2 (pY266)

Gab1 (pY285/307/317)

[Ana

lyte

] (pM

)

Time (min)

0.10 10 20 30 40 50 60 70

100

10

1

FAK (pY397/407) PLCg1 (pY1253)

pl3Kp110d (pY485) PKCm (pY502)

FAK (pY861)

[Ana

lyte

] (pM

)

Time (min)

0.010 10 20 30 40 50 60 70

1000

10

1

100

10

0.1

ERK1/2 (pY204/187) Csk (pY304)

STAT3 (pY705) SPRY2 (pY55)

Shc (pY349/350)

Figure 3. Phosphorylated EGFR pathway proteins, as well as total EGFR, were simultaneously detected in A431 cells treated with 100 ng/mL EGF. Lysates were collected at 0, 5, 19, 20, 30, 45 and 60 minutes. Values are internally normalized utilizing the TAFII68 loading control.

A

C

B

D

EpiQuant™ EGFR Panel: Gefitinib InhibitionFigure 4. Phosphorylated EGFR pathway proteins, as well as total EGFR, were simultaneously detected in A431 cells treated with gefitinib. Cells were pre-treated with gefitinib 15 minutes prior to the addition of 300 ng/mL EGF. Lysates were collected after 5 minutes EGF stimulation. Values are internally normalized utilizing the TAFII68 loading control.

[Ana

lyte

] (pM

)

[Gefitinib] (µM)

10.0001 0.001 0.01 0.1 1 10

10

100

1,000

10,000

EGFR total

EGFR (pY1110/1125) EGFR (pY1069/1092)

Shc (pY349/350)

Challenges in Advancing Cell Signaling ResearchTo 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 used to study 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 only 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 have enabled researchers to study phosphorylation levels of up to 12 proteins simultane-ously. 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, at best.

These issues have been addressed with the launch of Millipore’s MILLIPLEX® map EpiQuant™ technology. EpiQuant™ assays enable researchers to measure phosphorylation levels of multiple sites, as well as total protein, simultaneously with absolute quantitation. Based on the Luminex® xMAP® platform, the EpiQuant™ 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.

Features of Millipore’s MILLIPLEX® map EpiQuant™ AssaysThe complexity and number of protein targets involved in signaling events, as well as cellular responses, require tools that enable multiplex analysis of samples. Millipore’s MILLIPLEX® map EpiQuant™ technology enables for the analysis of a greater number of intracellular analytes per well, as 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, with lower coefficients of variation.

Our EpiQuant™ technology:

• Enables measurement of multiple phosphorylation sites simultaneously with picomolar sensitivities

• Enables absolute quantitation of multiple phospho-rylation sites on the same protein simultaneously

• Enables absolute quantitation of both total and phosphoproteins simultaneously

EpiQuant™ panels are the largest commercially available assays, enabling the user to:

• Customize an assay by selecting only the analytes of interest

• Design small- or large-plexed panels easily using one catalogue number

• Obtain absolute quantitative results at picomolar sensitivities

Ordering Information

Product Name Catalogue No.

MILLIPLEX® map EpiQuant™ Sample Preparation Kit (required with any EpiQuant™ panel) MPEQ-SP

MILLIPLEX® map EpiQuant™ EGFR Pathway Magnetic Bead Panel, 22-Plex MPEQMAG -110 K

MILLIPLEX® map EpiQuant™ EGFR Pathway Magnetic Bead Panel, 22-Plex Premix MPEQMG-110K-PX22

MILLIPLEX® map EpiQuant™ Phosphotyrosine Panel 1, 40-Plex MPEQ-100K

References

1. Yoshida, T; Zhang, G; Haura EB. Biochem Pharmacol. 2010, 80(5), 613-23.

2. Campbell, L; Blackhall, F; Thatcher, N. Expert Opin Pharmacother. 2010, 11(8),1343-57.

3. Lurje, G; Lenz, HJ. Oncology. 2009, 77(6), 400-10.

4. Nishida, K; Hirano, T. Cancer Sci. 2003, 94(12), 1029-33.

5. Pellegrini, M; Baldari, CT. Curr Mol Med. 2009, 9(3), 392-8.

6. Francia, P; Cosentino, F; Schiavoni, M; Huang, Y; Perna, E; Camici, GG; Lüscher, TF; Volpe, M. J Mol Med. 2009, 87(9), 885-91.

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EMD Millipore and the M logo are trademarks of Merck KGaA, Darmstadt, Germany.MILLIPLEX is a registered trademark of Millipore Corporation.MAPmates and EpiQuant are trademarks of Millipore Corporation.Luminex and xMAP are registered trademarks of Luminex Corporation.Luminex 200 is a trademark of Luminex Corporation.ATTC is registered trademark of American Type Culture Collection Corporation.Iressa is a registered trademark of AstraZeneca UK Limited.Lit No. TB3245EN00 Rev. A 11/2011 Job No. DP SBU-11-05427 Printed in the U.S.A.©2011 Millipore Corporation, Billerica, MA 01821 U.S.A. All rights reserved.

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