Sensitive multiplexed analysis of kinase activities and activity-based kinase identification

Abstract

Constitutive activation of one or more kinase signaling pathways is a hallmark of many cancers. Here we extend the previously described mass spectrometry–based KAYAK approach by monitoring kinase activities from multiple signaling pathways simultaneously. This improved single-reaction strategy, which quantifies the phosphorylation of 90 synthetic peptides in a single mass spectrometry run, is compatible with nanogram to microgram amounts of cell lysate. Furthermore, the approach enhances kinase monospecificity through substrate competition effects, faithfully reporting the signatures of many signaling pathways after mitogen stimulation or of basal pathway activation differences across a panel of well-studied cancer cell lines. Hierarchical clustering of activities from related experiments groups peptides phosphorylated by similar kinases together and, when combined with pathway alteration using pharmacological inhibitors, distinguishes underlying differences in potency, off-target effects and genetic backgrounds. Finally, we introduce a strategy to identify the kinase, and even associated protein complex members, responsible for phosphorylation events of interest.

Identification of Extracellular Signal-regulated Kinase 1 (ERK1) Direct Substrates using Stable Isotope Labeled Kinase Assay-Linked Phosphoproteomics

“…5A), cytoplasm, nucleus, membrane, and cytoskeleton, a distribution consistent with a previous report (40). In comparison with total human proteome (28,612 proteins) and phosphoproteome (11,479 proteins) having GO annotations from Uniprot and PhosphoSitePlus, respectively, the proportions of cytoplasm, nucleus, and cytoskeleton were significantly increased (Fig. 5B).…”

“…Mass spectrometry has been extensively used for kinasesubstrate interaction mapping (8) and global phosphorylation profiling (9). Although thousands of phosphorylation sites have been detected, complex phosphorylation cascade and crosstalk between pathways make it difficult for large-scale phosphoproteomics to reveal direct relationships between protein kinases and their substrates (10,11). Extensive statistics, bioinformatics, and downstream biochemical assays are mandatory for the substrate verification (12,13).…”

Identification of Bioactivating Enzymes Involved in the Hydrolysis of Laninamivir Octanoate, a Long-Acting Neuraminidase Inhibitor, in Human Pulmonary Tissue

“…Then, to seek proteins that showed a high correlation with LO-hydrolyzing activity, all proteins in the fractions were identified and quantified by LC-MS/MS analysis. Theoretically, protein of a higher correlation coefficient has higher probability of a responsible protein to the activity profile, and our previous experiences (Kubota et al, 2009;Sakurai et al, 2013, and unpublished results) are in agreement with this concept. In total, 2381 proteins were identified, but there was no single protein that had the bimodal chromatographic peaks highly correlated with the enzyme activity profile (data not shown).…”

“…is usually required to achieve the purification. On the other hand, we have recently extended an advanced methodology named proteomic correlation profiling to identify drug metabolizing enzymes (Sakurai et al, 2013), whose basic concept was previously reported by Kubota et al (2009). In this methodology, the biologic material is fractionated by column chromatography, followed by the calculation of each protein's correlation coefficient between the enzyme activity and proteomic profile of the fractions.…”

Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates

“…Large-scale phosphoproteomics, however, does not typically reveal precise connections between protein kinases and their direct substrates (11,12). In recent years, there have been increasing attempts to develop mass spectrometry-based proteomic strategies for the identification of elusive kinase substrates (7,13,14).…”