A prototype antibody microarray platform to monitor changes in protein tyrosine phosphorylation

Protein Center, and Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Molecular &amp Cellular Proteomics (Impact Factor: 6.56). 12/2004; 3(11):1102-18. DOI: 10.1074/mcp.M400075-MCP200
Source: PubMed


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.

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    • "Although many methodologies using proxies for kinase activities (e.g., antibody- or MS-based measurements of phosphorylation states upon biological perturbation) [30,31] are very useful and quantitative, these indirect inferences of kinase activity generally lack a temporal component, making the interpretation of enzymatic reaction rates very difficult. Additionally, other types of PTMs occurring on kinases as previously described play a role in modulating their activities, which are usually not accounted for in these studies. "
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    • "Flow cytometric analysis measuring both the bead color as well as the streptavidin R-phycoerythrin conjugate signal thus allows quantification of the relative (phospho)protein abundance. Inverse approaches encompassing an antibody microarray platform have been applied successfully as well, accommodating all of the previously mentioned advantages [110]. Although these strategies only allow detection of previously identified proteins and protein phosphorylation sites, a detailed picture of the phosphoproteome can still be obtained in a high-throughput fashion using limited specimen amounts. "
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    • "The array system takes a multiplex format and is able to detect cytokines to the picogramper-milliliter (pg/ml) level. Protein microarray has also been used to study protein–protein interactions, protein modifications, protein profiling, and enzymatic reactions [18] [19] [20] [21]. 0003-2697/$ -see front matter Ó 2010 Elsevier Inc. "
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