A prototype antibody microarray platform to monitor changes in protein tyrosine phosphorylation Journal Article
| Authors: | Gembitsky, D. S.; Lawlor, K.; Jacovina, A.; Yaneva, M.; Tempst, P. |
| Article Title: | A prototype antibody microarray platform to monitor changes in protein tyrosine phosphorylation |
| Abstract: | Reversible protein phosphorylation is a key regulatory process in all living cells. Deregulation of modification control mechanisms, especially in the case of tyrosine, may lead to malignant transformation and disease. Phosphotyrosine (p-Tyr) accounts for only 0.05% of the total cellular phospho-amino acid content, yet plays an unusually prominent role in eukaryotic signaling, development, and growth. Tracking temporal and positional p-Tyr changes across the cellular proteome, i.e. tyrosine phosphoproteomics, is therefore tremendously valuable. Here, we describe and evaluate a prototype antibody (Ab) microarray platform to monitor changes in protein Tyr phosphorylation. Availability permitting, a virtually unlimited number of Abs, each recognizing a specific cellular protein, may be arrayed on a chip, incubated with total cell or tissue extracts or with biological fluids, and then probed with a fluorescently labeled p-Tyr-specific monoclonal Ab, PY-KD1, specifically generated for this assay as part of the current study. The optimized protocol allowed detection of changes in the Tyr phosphorylation state of selected proteins using submicrogram to low nanogram of total protein extract, amounts that may conceivably be obtained from a thousand to a hundred thousand cells, or less, depending on the cell or tissue type. The assay platform was evaluated by assessing changes in a rationally selected subset of the Tyr phosphoproteome of Bcr-Abl-expressing cells treated with a specific inhibitor, Gleevec, and of epidermal growth factor (EGF)-treated HeLa cells. The results, ratiometric rather than strictly quantitative in nature, conformed with previous identifications of several Bcr-Abl and EGF receptor targets, and associated proteins, as detected by exhaustive mass spectrometric analyses. The Ab microarray method described here offers advantages of low sample and reagent consumption, scalability, detection multiplexing, and potential compatibility with microfluidic devices and automation. The system may hold particular promise for dissecting signaling pathways, molecular classification of tumors, and profiling of novel target-cancer drugs. © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. |
| Keywords: | signal transduction; epidermal growth factor; controlled study; protein expression; protein phosphorylation; protein array analysis; human cell; mass spectrometry; proteome; animals; cells, cultured; imatinib; epidermal growth factor receptor; fluorescent dye; fluorescent dyes; protein; clinical protocol; hela cell; hela cells; pyrimidines; phosphorylation; automation; assay; cell type; monoclonal antibody; antibodies, monoclonal; eukaryota; molecular recognition; device; development; protein-tyrosine kinases; bcr abl protein; piperazines; malignant transformation; tumor classification; protein microarray; eukaryote; body fluid; reagent; sample; phosphotyrosine; growth; incubation time; tissue types; tissue extract; humans; human; priority journal; article; phosphoamino acid |
| Journal Title: | Molecular & Cellular Proteomics |
| Volume: | 3 |
| Issue: | 11 |
| ISSN: | 1535-9476 |
| Publisher: | Amer Soc Biochemistry Molecular Biology Inc |
| Date Published: | 2004-11-01 |
| Start Page: | 1102 |
| End Page: | 1118 |
| Language: | English |
| DOI: | 10.1074/mcp.M400075-MCP200 |
| PROVIDER: | scopus |
| PUBMED: | 15358805 |
| DOI/URL: | |
| Notes: | Mol. Cell. Proteomics -- Cited By (since 1996):84 -- Export Date: 16 June 2014 -- CODEN: MCPOB -- Source: Scopus |
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