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Updated: Aug 22, 2026
Assessment of Resistance to Tyrosine Kinase Inhibitors by an Interrogation of Signal Transduction Pathways by Antibody Arrays
Published on: September 19, 2018
A prototype antibody microarray platform to monitor changes in protein tyrosine phosphorylation
Dmitry S Gembitsky1, Kevin Lawlor, Andrew Jacovina
1Protein Center, and Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
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.
Insights
This study introduces a novel antibody microarray platform for monitoring protein tyrosine phosphorylation changes. The assay is sensitive, requires minimal sample, and aids in dissecting signaling pathways and classifying tumors.
Area of Science:
- Biochemistry and Molecular Biology
- Cellular Signaling
- Proteomics
Background:
- Protein phosphorylation, particularly tyrosine phosphorylation (p-Tyr), is crucial for cellular regulation.
- Dysregulation of tyrosine phosphorylation is linked to diseases like cancer.
- Tracking p-Tyr dynamics is vital for understanding cellular processes and disease mechanisms.
Purpose of the Study:
- To develop and evaluate a prototype antibody microarray platform for monitoring protein tyrosine phosphorylation.
- To assess the platform's ability to detect changes in tyrosine phosphoproteome.
- To demonstrate the platform's utility in studying signaling pathways and disease-related targets.
Main Methods:
- Development of a prototype antibody microarray platform.
- Utilizing a specific p-Tyr monoclonal antibody (PY-KD1) for probing.
- Incubation of arrays with cell/tissue extracts and subsequent probing with labeled antibodies.
- Evaluation using Bcr-Abl-expressing cells treated with Gleevec and EGF-treated HeLa cells.
Main Results:
- The platform successfully detected changes in tyrosine phosphorylation states of selected proteins.
- The assay demonstrated high sensitivity, requiring low amounts of total protein extract.
- Results correlated with established findings from mass spectrometry analyses for known signaling targets.
Conclusions:
- The antibody microarray platform offers a sensitive, scalable, and efficient method for tyrosine phosphoproteomics.
- Advantages include low sample/reagent consumption and multiplexed detection.
- The platform shows promise for dissecting signaling pathways, tumor classification, and drug profiling.
