Contact spotting of protein microarrays coupled with spike-in of normalizer protein permits time-resolved analysis of ERBB receptor signaling.
Protein microarrays allow highly accurate comparison and quantification of numerous biological samples in parallel while requiring only little material. This qualifies protein arrays for systems biology and clinical research where only limited sample material is available, but a precise readout is required. With the introduction of signal normalization steps to monitor the drop size of manually contact-spotted RP protein arrays, the usefulness of normalizer proteins to ensure a high-throughput but inexpensive protein analysis was demonstrated. This approach was applied for the analysis of signaling through ERBB receptor activated kinases in the breast cancer cell line MCF-7. Activation of ERK1/2 and AKT by ERBB1 (EGFR), ERRB2 (HER2/neu), and ERBB3-4 was monitored in a time-resolved manner. Analysis of pathway activation by stimulation with epidermal growth factor and heregulin, or inhibition by blocking with gefitinib or herceptin allowed a characterization of the distinct signaling properties of the different ERBB receptor subtypes.
Löbke
Christian
C
Division of Molecular Genome Analysis, German Cancer Research Center Heidelberg, Heidelberg, Germany.
Laible
Mark
M
Rappl
Claudia
C
Ruschhaupt
Markus
M
Sahin
Ozgür
O
Arlt
Dorit
D
Wiemann
Stefan
S
Poustka
Annemarie
A
Sültmann
Holger
H
Korf
Ulrike
U
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D061067:Antibodies, Monoclonal, Humanized; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000032:analysis / Q000378:metabolism*; D000077156:Gefitinib; D005982:Glutathione Transferase / Q000378:metabolism*; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000032:analysis*; D048052:Mitogen-Activated Protein Kinase 3 / Q000032:analysis*; D040081:Protein Array Analysis; D051057:Proto-Oncogene Proteins c-akt / Q000032:analysis; D011799:Quinazolines / Q000494:pharmacology; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D066247:Receptor, ErbB-4; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012015:Reference Standards; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D000068878:Trastuzumab; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D000077156:Gefitinib; D005982:Glutathione Transferase; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D051057:Proto-Oncogene Proteins c-akt; D011799:Quinazolines; D018719:Erb-b2 Receptor Tyrosine Kinases; D066247:Receptor, ErbB-4; D011993:Recombinant Fusion Proteins; D000068878:Trastuzumab; C508053:ERBB2 protein, human; C587689:ERBB4 protein, human