Multiplexing the Quantitation of MAP Kinase Activities Using Differential Sensing

• DOI: 10.1021/jacs.1c12757
• Publication Date: 2022-02-23T16:22:15Z
• Authors: Lingyu Zeng; Tamer S. Kaoud; Diana Zamora-Oliv...; Amanda L. Bohanon; Yiru Li; Jacey R. Pridgen; Yakndara E. Ekpo; Deborah L. Zhuang; Jessica R. Nye; Mitchell Telles; Michelle Winkler; Sebastian Rivera; Federico Marini; Kevin N. Dalby; Eric V. Anslyn
• Keywords: 0301 basic medicine; 03 medical and health sciences; MAP Kinase Signaling System; Humans; Mitogen-Activated Protein Kinases; Phosphorylation; Peptides; differential sensing; mitogen-activated protein kinases (MAPKs); sulfonamido-oxine (SOX)-labeled peptides; chemometrics; partial least squares (PLS); Protein Kinases; p38 Mitogen-Activated Protein Kinases; 3. Good health

Protein kinases are therapeutic targets for many human diseases, but the lack of user-friendly quantitative assays limits the ability to follow the activities of numerous kinases at once (multiplexing). To develop such an assay, we report an array of sulfonamido-oxine (SOX)-labeled peptides showing cross-reactivity to different mitogen-activated protein kinases (MAPKs) for use in a differential sensing scheme. We first verified using linear discriminant analysis that the array could differentiate MAPK isoforms. Then, using principal component analysis, the array was optimized based on the discrimination imparted by each SOX-peptide. Next, the activity of individual MAPK families in ternary mixtures was quantified by support vector machine regression. Finally, we multiplexed the quantification of three MAPK families using partial least squares regression in A549 cell lysates, which has possible interference from other kinase classes. Thus, our method simultaneously quantifies the activity of multiple kinases. The technique could be applied to other protein kinase families and the monitoring of diseases.