Chemoproteomic profiling of kinases in live cells using electrophilic sulfonyl triazole probes

Sulfonyl-triazoles are a new class of electrophiles that mediate covalent reaction with tyrosine residues on proteins through sulfur-triazole exchange (SuTEx) chemistry. Recent studies demonstrate the broad utility and tunability of SuTEx chemistry for chemical proteomics and protein ligand discovery. Here, we present a strategy for mapping protein interaction networks of structurally complex binding elements using functionalized SuTEx probes. We show that the triazole leaving group (LG) can serve as a releasable linker for embedding hydrophobic fragments to direct molecular recognition while permitting efficient proteome-wide identification of binding sites in live cells. We synthesized a series of SuTEx probes functionalized with a lipid kinase fragment binder for discovery of ligandable tyrosines residing in catalytic and regulatory domains of protein and metabolic kinases in live cells. We performed competition studies with kinase inhibitors and substrates to demonstrate that probe binding is occurring in an activity-dependent manner. Our functional studies led to discovery of probe-modified sites within the C2 domain that were important for downregulation of protein kinase C-alpha in response to phorbol ester activation. Our proof of concept studies highlight the triazole LG of SuTEx probes as a traceless linker for locating protein binding sites targeted by complex recognition elements in live cells.


Introduction
Covalent probes are important tools for studying protein biology and for advancing translational discoveries. 1,2 Fragments and functionalized inhibitor molecules bearing electrophiles or photoreactive groups have been used for proteomic discovery of ligand sites that can be targeted for pharmacological control [3][4][5] and protein degradation. 6,7 A suite of chemistries for investigating cysteine-, 5,8-10 lysine-, [11][12][13][14] aspartate/glutamate-, 15,16 and tyrosine- [17][18][19][20][21][22] residues is providing new opportunities to develop ligands for perturbing protein function. [4][5][6][7]11,23 Covalent probes have also provided creative solutions for studying post-translational modications (PTM) including phosphorylation, 17 methylation, 24 crotonolyation, 25 and deimination. 26 We introduced sulfur-triazole exchange (SuTEx) chemistry as a new class of electrophiles for chemical proteomic applications 17 and fragment-based ligand discovery 27 (FBLD). The SuTEx reaction occurs through nucleophilic attack of the sulfur center to facilitate protein reaction preferentially on tyrosine residues in lysates and live cells. 17,27 In contrast with the uoride leaving group (LG) on SuFEx, 28,29 the addition of a triazolide LG on SuTEx molecules introduced additional opportunities for tuning activity of the sulfur electrophile. We recently expanded our reactivity studies to investigate adduct group (AG) and LG modications for activating nucleophilic substitution reactions at the sulfur center. We applied the reactivity principles gained from our FBLD studies to demonstrate the tunability of SuTEx for developing ligands to disrupt functional tyrosine sites on proteins. 27 Ligandable tyrosine sites could also be discovered by functionalizing existing fragments with the SuTEx electrophile and bioorthogonal reporter groups. The triazole group is well positioned for incorporating recognition elements because of its synthetic accessibility and absence from modied binding sites aer covalent reaction to simplify chemical proteomic investigations (Fig. 1). The latter feature is especially important for determining the site of binding using fragment binders of medium to high structural complexity, which typically produce covalent probe-peptide adducts that are difficult to identify by liquid chromatography-mass spectrometry (LC-MS). To circumvent these issues, specialized proteomic workows have been developed, for example, to understand LC-MS fragmentation mechanisms and increase condence in binding site identications of cysteine-directed covalent drugs. 30,31 Given the complexities of LC-MS compound fragmentation mechanisms, these approaches, while effective, will likely need to be customized for each covalent probe analyzed.
Here, we present an alternative strategy for mapping protein interactions of structurally complex binding elements. We used a hydrophobic fragment (RF001) derived from the serotonin receptor antagonist ritanserin to determine whether its secondary activity against kinases in vitro was reected in live cells. We synthesized a series of RF001-functionalized SuTEx probes to discover modied tyrosines on known targets of ritanserin including sites important for biochemical function of diacylglycerol kinase-alpha and inhibitor binding of the nonreceptor tyrosine kinase FER. We applied RF001-SuTEx probes to prole inhibitor and substrate binding across a suite of ligandable tyrosine sites located in catalytic and regulatory domains of >50 native kinases in live T cells to discover regions of the C2 domain important for downregulating protein kinase C-alpha during prolonged cellular activation.

Results and discussion
Synthesis of RF001-functionalized SuTEx probes RF001 is a fragment derived from the serotonin receptor antagonist ritanserin that was tested in the clinic for treatment of psychiatric disorders. 32 Further investigations into ritanserin activity revealed secondary activity against DGKa 33 and other kinases. 34 RF001 showed improved specicity for DGKa compared with ritanserin as determined by activity-based proling studies using ATP acyl phosphate probes. 35 The proteome-wide activity of RF001, however, is currently unknown and is important for evaluating whether RF001 is a suitable fragment for developing kinase probes and inhibitors.
We modied the triazole group with the RF001 recognition element to produce functionalized SuTEx probes for evaluation of proteome activity. We chose to incorporate the RF001 moiety into the LG and not the AG for our probe design to avoid generating bulky probe-peptide adducts that could complicate LC-MS site-of-binding studies. We developed a synthetic route to incorporate RF001 as a recognition element for functionalized SuTEx probe design. Specically, ethyl N-Boc-piperidine-4carboxylate was reacted with two equivalents of 4-uorophenyl magnesium bromide to produce an intermediate that was dehydrated and deprotected with TFA in one pot to form the RF001 product (Scheme 1). RF001 was synthesized with excellent yield (>80%) without the need for additional column chromatography. A terminal alkyne was installed through alkylation of the piperidine nitrogen. Compound TH207 was synthesized following a copper(I) thiophene-2-carboxylate (CuTC)-mediated coupling 36 between the RF001-alkyne intermediate and tosyl azide with moderate yield (50%). Next, the tosyl group was removed and the resulting triazole intermediate coupled to various alkyne-containing sulfonyl chlorides to produce the corresponding SuTEx probes. See ESI † for synthetic procedures and probe characterization.

Adduct group modications tune reactivity of RF001-SuTEx probes in proteomes
We used a gel-based chemical proteomic assay to evaluate reactivity of RF001-SuTEx probes. Each SuTEx probe shared a common RF001-modied triazole group but differed by the chemical linker for attachment of the alkyne reporter tag. The alkyne group was incorporated into the AG structure through an amide (TH211), methoxy (TH214), or direct conjugation (TH216) to the phenyl of the sulfonyl group ( Fig. 2A). Considering the sensitivity of SuTEx reaction to AG modications, 27 we reasoned that these structurally analogous probes would show differences in proteome reactivity and provide further insights into electronic effects for tuning activity of the sulfur electrophile.
To compare proteome activity of RF001-SuTEx probes, Jurkat soluble and membrane proteomes were treated with TH211, TH214, or TH216 (100 mM, 1 h, 37 C) followed by coppercatalyzed azide-alkyne cycloaddition (CuAAC 37 ) conjugation of rhodamine-azide to visualize probe-labeled protein targets resolved by SDS-PAGE and detected by in-gel uorescence scanning. Our gel-based experiments identied TH211 as the most reactive probe as evidenced by robust uorescence labeling of proteins across the entire molecular weight range (Fig. 2B). These ndings are in agreement with our previous studies that demonstrated modications of the AG with electron-withdrawing groups (e.g. the carbonyl of TH211) can enhance reactivity of SuTEx reaction in solution and proteomes. 27 This reactivity prole was also supported by differences in activity for TH214 and TH216. Although both probes showed reduced labeling compared with TH211, the electron donating character of the alkoxy group 38 of TH214 could help explain the decrease in reactivity compared with TH216 ( Fig. 2B). Gel-based analyses of TH211-, TH214-and TH216treated HEK293T proteomes yielded reactivity proles similar to those observed with Jurkat proteomes (Fig. S1 †).
In summary, we utilized gel-based chemical proteomics to compare reactivity of SuTEx probes bearing a common RF001-modied LG and differentiated by the reporter tag conjugation to the AG. We demonstrate that changing the chemical connectivity of the alkyne tag on the AG can profoundly affect proteome reactivity of resulting RF001-SuTEx probes.

RF001 binding element directs molecular recognition of SuTEx probes in proteomes
Next, we deployed quantitative chemical proteomics 17,27 to evaluate proteome-wide activity of RF001-SuTEx probes. We performed chemical proteomic studies using recombinant DGKa overexpressed lysates for proof of concept that the RF001 binding element was directing molecular recognition. In support of RF001-mediated binding, we observed concentration-dependent labeling of an $80 kDa uorescent TH211-labeled band in recombinant FLAG-tagged rat DGKa (rDGKa) overexpressed but not mock transfected HEK293T membrane and soluble proteomes (Fig. S2 †). Next, we analyzed isotopically light and heavy amino acid-labeled recombinant DGKa-HEK293T proteomes to enable quantitative LC-MS by SILAC. 39 Here, we focused our efforts on human DGKa (hDGKa) because the RF001 binding sites against the human protein have not yet been identied. In brief, recombinant light and heavy hDGKa-HEK293T cell proteomes were treated with RF001-SuTEx probes (100 mM, 1 h, 37 C) or dimethyl sulfoxide (DMSO) vehicle, respectively, followed by CuAAC 37 coupling with a desthiobiotin-azide tag. Proteomes were digested with trypsin protease and desthiobiotin-modied peptides enriched by avidin affinity chromatography, released, and analyzed by high-resolution LC-MS as previously described 17,27 and depicted in Fig. 1. Probe-modied peptide-spectrum matches (PSMs) that met our quality control condence criteria of $300 Byonic score, 40 1% protein false discovery rate (FDR), and #5 ppm mass accuracy were selected for further evaluation to minimize false positives. 17 SILAC ratios (SR) from these chemical proteomic studies (light -SuTEx probe/heavy -DMSO vehicle) were used to identify probe-modied peptides that were substantially enriched in probe-versus vehicle-treated samples (SR >5). We compared reactivity proles of each respective RF001-SuTEx probe across >3000 distinct probe-modied sites from $1200 detected proteins (Fig. 2C). In agreement with our gel-based results ( Fig. 2B and S1 †), we observed an approximate 2-4-fold higher number of TH211-compared with TH214-or TH216-modied tyrosine sites ($2-4-fold higher number of modied sites, Fig. 2C). The difference in reactivity between TH214 and TH216 was also recapitulated in our quantitative proteomic experiments with the latter SuTEx probe showing a >2-fold enhanced reactivity in the number of modied sites and proteins (Fig. 2C). A complete list of in vitro RF001-SuTEx probe-modied sites can be found in Table S1. † We showed in previous studies that RF001 engages the rDGKa active site through interactions with the catalytic domain 35 and C1 lipid recognition domain. 41 Here, we reasoned that probe-modied binding sites identied consistently across all 3 RF001-SuTEx probes would represent hDGKa active site regions that have a high propensity for ligand binding. In support of our hypothesis, we identied probe-modied sites in several domains, which we previously identied as ligandbinding regions of the rat DGKa active site using ATP acyl phosphate activity-based probes of kinases. 35 Specically, we identied probe binding at the C1A (Y240, Y258), DAGKc (Y399, Y477), and DAGKa (Y623, Y669) domains of recombinant hDGKa that showed specic enrichment across all 3 RF001-SuTEx probes in our chemical proteomic studies (SR >5, In summary, we demonstrate that installment of a RF001 binding element on SuTEx probes can direct molecular recognition to the hDGKa active site and reveal ligandable tyrosine sites within the C1A and DAGKc/DAGKa domains. In contrast with ATP acyl phosphate probes used previously, the RF001-SuTEx probes identied novel binding sites within the RVH and EF-hand domains of DGKa. Future studies will focus on evaluating the biochemical and metabolic impact of perturbing these DGKa tyrosine sites.

Live cell proling of inhibitor binding interactions against native DGKa and FER
Next, we deployed quantitative chemical proteomics 17,27 to map TH211 binding sites in live Jurkat T cells cultured in SILAC media. We treated Jurkat cells with varying concentrations of TH211 followed by gel-based chemical proteomic analysis to determine the optimal probe concentration for our quantitative  Table S1. † All data shown are representative of 3 experiments (n ¼ 3 biologically independent experiments).
LC-MS studies. We observed concentration-dependent probe labeling of proteomes from TH211-treated Jurkat cells and identied a saturating probe concentration for our LC-MS studies (50 mM TH211, Fig. S4 †).
Next, light and heavy SILAC Jurkat cells were treated with TH211 probe (50 mM, 2 h) or DMSO vehicle, respectively, followed by cell lysis and quantitative chemical proteomics by LC-MS (Fig. 1). Initially, we evaluated whether TH211 could mediate molecular recognition and binding to native hDGKa in live cells. These studies are important because current probes for DGKs are not suitable for direct activity-based proling in live cells. We identied prominent TH211 labeling of hDGKa in regions that demonstrated specic enrichment (SR >5) of active site binding of TH211 in live cells (Fig. 3A). Specically, we identied TH211 modied sites within the C1A (Y240) and accessory domain (Y544, Y623) of native hDGKa (Fig. 3B). We showed that the Y240F mutant is catalytically impaired compared with wild-type protein, which supports the ability of TH211 to bind functional sites important for hDGKa activity (Fig. S5 †). Interestingly, the binding site proles were dependent to some degree on the fraction analyzed; certain probe-modied peptides were detected preferentially in soluble, membrane, or both fractions (see ESI Methods for details †). Additional studies are needed to determine whether these differences in live cell probe labeling reect differential regulation of hDGKa (e.g. autoinhibition) in cellular environments.
Having established the TH211 binding prole for hDGKa, we performed competition studies using reported DGKa inhibitors (ritanserin) and broad-spectrum kinase inhibitors (staurosporine) to determine whether TH211-hDGKa interactions in  5 mM), respectively, for 1 h followed by TH211 probe labeling (50 mM) for 2 h. Cells were lysed and subjected to LC-MS chemical proteomics as described in Methods. The degree of inhibition of probe labeling by compounds at respective sites was quantified by the SR of light to heavy MS1 peptide abundances. The heatmap depicts sensitivity of tyrosine sites on DGKa and FER to compound treatments in order to determine site of binding for inhibitors tested. A complete list of SuTEx probe-modified sites and proteins from live cells studies can be found in Table S1. † All data shown are representative of n ¼ 2-3 biologically independent experiments. situ are functionally relevant. RF001 was not used as the competitor because this fragment compound requires millimolar concentrations 35 for effective binding to target proteins and this high amount of compound is not suitable for live cell studies. Light and heavy SILAC Jurkat cells were treated with vehicle or inhibitor (ritanserin, 25 mM; staurosporine, 1 or 0.5 mM; 60 min at 37 C) followed by TH211 probe labeling (50 mM, 2 h). Cells were lysed and processed for quantitative chemical proteomic analysis to evaluate target engagement of competitors at respective hDGKa binding sites. We observed mild competition (SR $2) at the C1 domain (Y240) and DAGKa catalytic domain (Y623) in ritanserin-treated cells, which is in agreement with previous observation of ritanserin activity against rat DGKa 35 (Fig. 3C). The other TH211-modied sites of hDGKa did not appear to be inhibited by ritanserin treatments (Fig. 3C).
While ritanserin showed mild activity against hDGKa, we observed potent activity of this compound against FER kinase in live cells as determined by blockade of TH211 probe labeling at the Y714 site (SR $4, Fig. 3C). These ndings are in agreement with our previous ATP acyl phosphate kinome proling studies that identied FER as the principal kinase targeted by ritanserin. 35 Treatment of Jurkat cells with the broad-spectrum kinase inhibitor staurosporine resulted in concentrationdependent blockade of TH211 probe labeling at FER Y714 (SR of 2 and 5 for 0.5 and 1 mM staurosporine, respectively; Fig. 3C). These ndings are in agreement with previous reports that staurosporine is competitive for active site probe labeling of FER. 43 Interestingly, we also observed competition at the Y544 site of hDGKa in Jurkat cells treated with staurosporine (Fig. 3C).
In summary, our chemical proteomic ndings demonstrate the utility of SuTEx probes such as TH211 that exhibit broad reactivity and binding recognition for mapping protein targets directly in live cells (Fig. 3 and Table S1 †). We identify ligandbinding sites on native hDGKa in domain regions that are in  Table S1 † for complete list of TH211-modified sites on kinases from live Jurkat cell studies. The protein kinase tree was generated using KinMap as previously described. 63 Kinome tree illustration reproduced courtesy of Cell Signaling Technology, Inc. (http://www.cellsignal.com). The lipid kinase tree was generated in-house as previously described. 34 All data shown are representative of 3 experiments (n ¼ 3 biologically independent experiments). agreement with previous chemoproteomic evaluation of DGK active sites. We also demonstrate that TH211 can be used to evaluate inhibitor binding against lipid (DGKa) and protein (FER) kinases directly in live cells to support activity proles previously reported in vitro.

TH211 enables identication of functional tyrosine sites on kinases
Closer inspection of our Jurkat data revealed a substantial number of kinases modied by TH211 in live cells (>50 kinases, Fig. 4; see ESI † for details of kinase analysis). The majority of probe-modied kinases mediate phosphorylation of protein substrates ( Fig. 4 and Table 1). We performed a Reactome pathway enrichment analysis to gain further insights into molecular pathways that are overrepresented among kinases in our TH211-modied kinase dataset 44 (Fig. 5A and S6 †). From this global analysis, we identied enrichment in several pathways involved in signal transduction including mitogenactivated protein kinase (MAPK) signaling and activation (signaling by RAFs, MAPK signaling/activation; Fig. 5A). A common mediator of these signaling pathways are the extracellular signal-regulated kinases (ERK1 and ERK2) that regulate proliferation, differentiation, apoptosis, and migration as part of the MAPK pathway. [45][46][47] In contrast to our previous chemoproteomic studies to map ATP binding sites of ERK isoforms, 48 we identied TH211-modied tyrosines in the F-site recruitment site (FRS) of ERK2 (Y263) involved in binding substrates that contain a conserved Phe-X-Phe-Pro consensus sequence docking site 47,49,50 (DEF motif, Fig. 5B). We also identied a homologous TH211-modied site on ERK1 (Y280). We performed competition studies in vitro with free ATP (1 mM) to show moderate competition ($50%, SR $2), which supports  functional binding from TH211 at these ERK sites ( Fig. 5C; see ESI Methods † for details of ATP competition assay). Given that the FRS is only accessible in the active phosphorylated ERK1/ 2, 47 our ndings identify ligandable tyrosines for blocking FRS protein-protein interactions in future medicinal chemistry efforts. The full list of TH211-modied binding sites and corresponding kinases can be found in Table S1. † Although the majority of TH211 kinase targets were assigned to the protein kinase class, we observed a substantial fraction of modied kinases involved in phosphorylation of metabolites ($40% of all detected kinases; Fig. 4 and Table S1 †). In addition to DGKa, we identied several TH211-modied lipid kinases involved in phosphorylation of phosphatidylinositol (PI)phosphate analogs to produce the secondary messenger PIP2 (ref. 51) (PIP5K1C, PIP5K1A, PIP4K2C; Fig. 4). We also identied a TH211-modied binding site (Y465) in the C-lobe of the catalytic domain of PI4K2A that is important for membrane association 52 (Fig. 4 and Table S1 †). In addition to phospholipid metabolism, we identied TH211-modied sites near the DAGKc domain (Y224) of the lipid kinase AGK, which is involved in phosphorylation of glycerol lipids 53 ( Fig. 4 and Table  1). The remaining probe modied-kinases largely mediate phosphorylation in glycolysis and metabolic pathways of nucleotides and sugars (Reactome pathways enrichment analysis of non-protein kinases, Fig. S7 †).
An enabling feature of our approach is to identify new ligand sites on protein kinases that may not be amenable to discovery with traditional biochemical and ATP-based chemoproteomic tools. Considering our TH211 studies are performed in situ, we have an opportunity to map ligand binding sites of kinases that may only be accessible and detected in live cell environments. These studies could also reveal ligand binding sites outside of the catalytic domain. We performed a domain enrichment analysis on probe-modied sites 17 to evaluate domains within kinases that are targeted by TH211 (Fig. 6A and Table 1). Interestingly, we identied non-catalytic domains that showed preferential, albeit with varying degrees of statistical signicance, enrichment with TH211. Several of these regulatory domains have discrete roles in regulating and localizing the activity of the catalytic domain of kinases. 54 We performed ATP competition studies in Jurkat lysates to determine whether TH211 binding to kinase sites was activity dependent. We identied a suite of kinase tyrosine sites that were sensitive to blockade of probe labeling with free ATP (Fig. 6B). We tested different concentrations of ATP to evaluate concentration dependence, which can in some instances help localize the ATP binding region of kinase active sites. For example, we compared sensitivity of Y506 and Y164 on ZAP70 kinase and found that the former probe-modied site was potently competed at both ATP concentrations tested (1 and 5 mM free ATP, Fig. 6B). These ndings are in agreement with crystal structures showing that Y506 is localized in the ATPbinding pocket and in direct proximity to an ATP analog (PDB ID: 4K2R, Fig. 6C). The reduced sensitivity of Y164 to ATP competition is in agreement with the location of this site in the SH2 domain of ZAP70 (Y164, Fig. 6B). T cell activation leads to TCR phosphorylation, which provides docking signals for SH2mediated recruitment of ZAP70 and subsequent phosphorylation and activation of this kinase. 55 The identication of a tyrosine-modied site (Y164) in vicinity of the phospho-recognition site of ZAP70 SH2 domain presents an opportunity in future studies to inactivate ZAP70 via blockade of its localization to the TCR.
We identied additional examples of functional TH211 binding to kinases by comparing ATP-competed sites in our chemical proteomic assay to their respective active site location in crystal structures. For example, the Y233 site of MAP2K2 (MEK2) kinase showed concentration-dependent blockade of probe labeling in lysates treated with free ATP (SR of 2 and 6 for 1 and 5 mM ATP, Fig. 6B). Co-crystal structures of MEK2 with ATP substrate 56 veried our chemical proteomic ndings by showing Y233 in close proximity to substrate in the MEK2 active site (PDB ID: 1S9I, Fig. 6C). We also identied probe-modied tyrosines in active site regions of MAPK14 and MAPKAPK3 that mediate binding to reported inhibitors. The sensitivity of Y182 and Y76 to ATP competition are in agreement with location of these tyrosine sites in ATP-binding regions of MAPK14 (ref. 57) (PDB ID: 4EWQ) and MAPKAPK3 (ref. 58) (PDB ID: 3SHE, Fig. 6C).
In summary, we demonstrate that TH211 is suitable for live cell chemoproteomic proling of protein and metabolic kinases to reveal tyrosine (and lysine) sites in catalytic and regulatory domains of kinases. Identication of ligandable tyrosine sites in ATP and inhibitor binding regions of kinases provides future opportunities for developing covalent inhibitors against these key signaling proteins.
Identication of C2 domain sites important for downregulation of PKC-a in response to PMA Among candidate sites in kinase regulatory domains, we selected tyrosine-(Y195) and lysine-modied sites (K209, K232) in the C2 domain of protein kinase C-alpha for further studies (PRKCA or PKC-a, Table 1). The C2 domain is important for regulating activation of PKC-a-mediated signaling in cells. 59 To test the function of these probe-modied sites, we performed mutagenesis studies to evaluate response of recombinant wildtype and mutant PKC-a proteins to cell activation with the DAG mimetic phorbol-12-myristate-13-acetate (PMA 60 ). Specically, prolonged activation with PMA triggers degradation of phorbol ester-responsive PKC isoforms to downregulate cellular responses. 61 Consistent with previous reports, we observed substantial loss of PKC-a in recombinantly overexpressing HEK293T cells treated with PMA under chronic activation conditions (500 nM PMA, 6 h; Fig. 7A and S8 †). Evaluation of PKC-a mutant response under the same activation paradigm showed equivalent downregulation ($88% loss of protein) for the Y195F mutant protein. In contrast, PKC-a K209A mutant showed a signicantly blunted response compared with its wild-type counterpart ($68%, Fig. 7B). The observed differences in downregulation of PKC-a wild-type and mutant proteins were not due to expression because basal (control) recombinant protein levels were comparable with the exception of K232A, which showed signicantly reduced control and PMAstimulated protein levels (Fig. 7B). Sequence homology analyses revealed that K209 of PKC-a is evolutionarily conserved and further supports the functional importance of this probe modied amino acid ( Fig. 7C and S9 †).
Collectively, our studies support the ability of TH211 to identify functional sites in the C2 domain of PKC-a that are important for downregulation during chronic stimulation with PMA. Future studies will focus on determining whether liganding C2 domain sites can be utilized for pharmacological modulation of PKC-a levels and function in activated cells.

Conclusion
Here, we demonstrate that a distinct advantage of the SuTEx electrophile is the capability for introducing structurally complex binding elements into the LG for directing molecular recognition on proteins without compromising the ability to map probe-modied sites by LC-MS chemical proteomics. The triazolide LG of SuTEx probes is well-suited as a traceless linker in LC-MS studies to expedite direct proteome-wide identication of binding sites in living systems. As proof of concept, we combined an existing lipid kinase fragment binding element with the SuTEx electrophile and bioorthogonal reporter tags to produce a kinase covalent probe (TH211) suitable for lysate and live cell chemoproteomic proling (Fig. 4). We evaluated TH211 activity in live Jurkat T cells to identify modied sites in ATPand lipid-substrate binding regions of DGKa, which matched previous chemical proteomics studies using orthogonal ATPbased probes (Fig. 3). Our nding that mutation of a probe-modied site in the C1 domain results in loss of catalytic activity of human DGKa helps support functional TH211 binding (Fig. S5 †).
TH211 activity was evaluated across the kinome to identify additional metabolic and protein kinases that contained tyrosine and lysine sites amenable to SuTEx reaction ( Fig. 4 and Table 1). Importantly, we demonstrate that TH211-binding to these kinase targets in lysates and live cells is activity dependent and can be blocked using ATP substrate and kinase inhibitors ( Fig. 3 and 6). Interestingly, we identied TH211-modied sites in the C2 domain of PKC-a and demonstrated by site-directed mutagenesis that perturbations to a lysine site (K209) can affect downregulation of PKC-a upon prolonged exposure to PMA activation (Fig. 7). Additional follow-up studies are needed to determine the mechanism of PMA-induced degradation of PKC-a C2 domain mutants (e.g. proteasome-dependent or -independent pathways) and whether PKC-a protein function can be controlled through liganding of C2 domain sites using selective SuTEx compounds.
Another outcome from our LC-MS chemical proteomic studies was the inability to detect TH211-modied peptides that corresponded to any of the known human serotonin receptors (5-HTRs). These ndings were somewhat surprising given that RF001 is derived from ritanserin, which is a potent inverse agonist of 5-HTR. 62 Several factors could contribute to the lack of identication of peptides from 5-HTRs including low abundance, activation state, and probe-modied peptides that are not LC-MS compatible. Future studies evaluating additional cell types and LC-MS method are likely needed to conclusively determine whether RF001-based SuTEx probes engage these GPCRs.
In summary, we envision our chemoproteomic strategy can be generally extended to streamline LC-MS identication of binding sites of drug molecules and other chemically complex compounds, which is an important step towards understanding mode of action of small molecules.

Methods
Detailed Methods are provided in the ESI †

Conflicts of interest
There are no conicts to declare.