Multiplexing the Quantitation of MAP Kinase Activities Using Differential Sensing.

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.

Quantification of ERK Kinase Activity in Biological Samples Using Differential Sensing.

The understanding of complex biological systems requires an ability to evaluate interacting networks of genes, proteins, and cellular reactions. Enabling technologies that support the rapid quantification of these networks will facilitate the development of biological models and help to identify treatment targets and to assess treatment plans. The biochemical process of protein phosphorylation, which underlies almost all aspects of cell signaling, is typically evaluated by immunoblotting procedures (Western blot) or more recently proteomics procedures, which provide qualitative estimates of the concentration of proteins and their modifications in cells. However, protein modifications are difficult to correlate with activity, and while immunoblotting and proteomics approaches have the potential to be quantitative, they require a complex series of steps that diminish reproducibility. Here, a complementary approach is presented that allows for the rapid quantification of a protein kinase activity in cell lysates and tumor samples. Using the activity of cellular ERK (extracellular signal-regulated kinase) as a test case, an array sensing approach that utilizes a library of differential peptide-based biosensors and chemometric tools was used to rapidly quantify nanograms of active ERK in micrograms of unfractionated cell lysates and tumor extracts. This approach has the potential both for high-throughput and for quantifying the activities of multiple protein kinases in a single biological sample. The critical advantages of this differential sensing approach over others are that it removes the need for the addition of exogenous inhibitors to suppress the activities of major off-target kinases and allows us to quantitate the amount of active kinase in tested samples rather than measuring the changes in its activity upon induction or inhibition.