Summary Protein kinases are intensely studied mediators of cellular signaling, yet important questions remain regarding their regulation and in vivo properties. Here we use a probe-based chemoprotemics platform to profile several well studied kinase inhibitors against more than 200 kinases in native cell proteomes and reveal new biological targets for some of these inhibitors. Several striking differences were identified between native and recombinant kinase inhibitory profiles, in particular, for the Raf kinases. The native kinase binding profiles presented here closely mirror the cellular activity of these inhibitors, even when the inhibition profiles differ dramatically from recombinant assay results. Additionally, Raf activation events could be detected upon live cell treatment with inhibitors. These studies highlight the complexities of protein kinase behavior in the cellular context and demonstrate that profiling with only recombinant/purified enzymes can be misleading.

Caspase proteases are principal mediators of apoptosis, where they cleave hundreds of proteins. Phosphorylation also plays an important role in apoptosis, although the extent to which proteolytic and phosphorylation pathways crosstalk during programmed cell death remains poorly understood. Using a quantitative proteomic platform that integrates phosphorylation sites into the topographical maps of proteins, we identify a cohort of over 500 apoptosis-specific phosphorylation events and show that they are enriched on cleaved proteins and clustered around sites of caspase proteolysis. We find that caspase cleavage can expose new sites for phosphorylation, and, conversely, that phosphorylation at the +3 position of cleavage sites can directly promote substrate proteolysis by caspase-8. This study provides a global portrait of the apoptotic phosphoproteome, revealing heretofore unrecognized forms of functional crosstalk between phosphorylation and caspase proteolytic pathways that lead to enhanced rates of protein cleavage and the unveiling of new sites for phosphorylation.

Characterization and functional annotation of the large number of proteins predicted from genome sequencing projects poses a major scientific challenge. Whereas several proteomics techniques have been developed to quantify the abundance of proteins, these methods provide little information regarding protein function. Here, we present a gel-free platform that permits ultrasensitive, quantitative, and high-resolution analyses of protein activities in proteomes, including highly problematic samples such as undiluted plasma. We demonstrate the value of this platform for the discovery of both disease-related enzyme activities and specific inhibitors that target these proteins.capillary electrophoresis ͉ fluorescence ͉ MS ͉ protease T he completion of several major genome sequencing projects has both accelerated the pace and broadened the scale of modern biology. For example, it is estimated that a complete molecular understanding of the human organism will require the characterization of at least 100,000 proteins (1). Several innovations in the field of proteomics have advanced to meet the demands of postgenome biology. Particularly, improvements in the resolving capacity and reproducibility of 2D gel electrophoresis (2) and the increased sensitivity and accuracy of modern MS methods (3) have enabled the global characterization of protein expression levels in complex biological systems. Nonetheless, the large dynamic range of protein expression in biological tissues and fluids renders these methods ineffective at detecting and͞or quantitating low-abundance proteins in the absence of labor-intensive prefractionation or enrichment procedures (2, 4). Moreover, abundance-based proteomic strategies fail to provide direct information regarding protein function or activity.To address these limitations, a chemical proteomic technology called activity-based protein profiling (ABPP) (5) has emerged that utilizes active site-directed probes to enable the parallel measurement of the activity of many enzymes in complex proteomic mixtures (5-8). Activity-based probes (ABPs) enable the detection of changes in enzyme activity independent of alterations in protein abundance, and provide a specific enrichment strategy for low-copy-number enzymes without interference from more abundant proteins. To date, ABPP has relied on gel-based methods for proteome analysis that are difficult to automate and often fail to resolve highly related protein species (5, 6, 9, 10). Here, we describe a gel-free platform for ABPP, termed Xsite, that permits the rapid and systematic quantification and identification of enzyme activities in complex protein mixtures. Materials and MethodsGeneral Materials and Methods. Fluorophosphonate probes were synthesized as described (5). Synthesis of the cathepsin probe, AX6429, is detailed in supplemental materials (Fig. 3, which is published as supporting information on the PNAS web site). Rat FAAH was expressed in Escherichia coli and purified as described (11). Porcine trypsin and equine butyrylcholinesterase were p...

We have examined the effects of dissolved molecular oxygen on multiphoton-excited (MPE) photochemical derivatization of serotonin (5HT) and related cellular metabolites in various buffer systems and find that oxygen has a profound effect on the formation efficiency of visible-emitting photoproducts. Previously, end-column MPE photoderivatization provided low mass detection limits for capillary electrophoretic analysis of hydroxyindoles, but relied on the use of Good's buffers to generate high-sensitivity visible signal. In the present studies, visible emission from 5HT photoderivatized in different buffers varied by 20-fold under ambient oxygen levels but less than 2-fold in the absence of oxygen; oxygen did not significantly alter the photoproduct excited-state lifetime (approximately 0.8 ns). These results support a model in which oxygen interferes with formation of visible-emitting photoproducts by quenching a reaction intermediate, an effect that can be suppressed by buffer molecules. Deoxygenation of capillary electrophoresis separation buffers improves mass detection limits for 5-hydroxyindoles fractionated in 600-nm channels by approximately 2-fold to < or =30000 molecules and provides new flexibility in identifying separation conditions for resolving 5HT from molecules with similar electrophoretic mobilities, such as the catecholamine neurotransmitters.

The interaction of a drug with its target is critical to achieve drug efficacy. In cases where cellular environment influences target engagement, differences between individuals and cell types present a challenge for a priori prediction of drug efficacy. As such, characterization of environments conducive to achieving the desired pharmacologic outcome is warranted. We recently reported that the clinical CDK4/6 inhibitor palbociclib displays cell type-specific target engagement: Palbociclib engaged CDK4 in cells biologically sensitive to the drug, but not in biologically insensitive cells. Here, we report a molecular explanation for this phenomenon. Palbociclib target engagement is determined by the interaction of CDK4 with CDKN2A, a physiologically relevant protein inhibitor of CDK4. Because both the drug and CDKN2A prevent CDK4 kinase activity, discrimination between these modes of inhi-bition is not possible by traditional kinase assays. Here, we describe a chemo-proteomics approach that demonstrates high CDK4 target engagement by palbociclib in cells without functional CDKN2A and attenuated target engagement when CDKN2A (or related CDKN2/INK4 family proteins) is abundant. Analysis of biological sensitivity in engineered isogenic cells with low or absent CDKN2A and of a panel of previously characterized cell lines indicates that high levels of CDKN2A predict insensitivity to palbociclib, whereas low levels do not correlate with sensitivity. Therefore, high CDKN2A may provide a useful biomarker to exclude patients from CDK4/6 inhibitor therapy. This work exemplifies modulation of kinase target engagement by endogenous proteinaceous regulators and highlights the importance of cellular context in predicting inhibitor efficacy.