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Multiplexing the Quantitation of MAP Kinase Activities Using Differential Sensing.
Lingyu Zeng1, Tamer S Kaoud2, Diana Zamora-Olivares1,3
1Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, United States.
Journal of the American Chemical Society
|February 23, 2022
Summary
This study introduces a novel assay for simultaneously quantifying multiple protein kinase activities. The method uses labeled peptides and advanced data analysis to monitor kinase function in complex biological samples, aiding disease research.
Area of Science:
- Biochemistry
- Molecular Biology
- Analytical Chemistry
Background:
- Protein kinases are crucial therapeutic targets for various human diseases.
- Current quantitative assays lack user-friendliness and multiplexing capabilities for simultaneous kinase activity monitoring.
- Mitogen-activated protein kinases (MAPKs) play significant roles in cellular signaling pathways.
Purpose of the Study:
- To develop a user-friendly, multiplexed quantitative assay for monitoring protein kinase activities.
- To create an array of sulfonamido-oxine (SOX)-labeled peptides for differential sensing of MAPKs.
- To enable simultaneous quantification of multiple kinase activities in complex biological matrices.
Main Methods:
- Development of a sulfonamido-oxine (SOX)-labeled peptide array for differential sensing.
- Application of linear discriminant analysis (LDA) for differentiating MAPK isoforms.
- Optimization of the array using principal component analysis (PCA).
- Quantification of individual MAPK families in mixtures using support vector machine (SVM) regression.
- Multiplexed quantification of three MAPK families in A549 cell lysates using partial least squares (PLS) regression.
Main Results:
- The SOX-peptide array successfully differentiated MAPK isoforms using LDA.
- PCA was used to optimize the array based on peptide discrimination.
- SVM regression accurately quantified individual MAPK families in ternary mixtures.
- PLS regression enabled multiplexed quantification of three MAPK families in cell lysates, overcoming potential interference.
- The assay demonstrated simultaneous quantification of multiple kinase activities.
Conclusions:
- The developed SOX-peptide array assay enables simultaneous quantification of multiple protein kinase activities.
- This technique offers a user-friendly and multiplexed approach for kinase activity monitoring.
- The method has potential applications in studying other protein kinase families and monitoring disease progression.
