in target - based approaches 
 to drug discovery , linking the observed 
 phenotypic response to a ligand of interest with on - target modulation 
 is a critical step . to this end ,
both 
 on- and off - targets of a drug candidate need to be identified and 
 characterized prior to clinical development . among many target identification 
 methods ,
photoaffinity - labeling is particularly attractive , as the 
 transient association of the molecular targets with a drug candidate 
 becomes permanent after photo - cross - linking in the native cellular 
 environment . in addition , for targets that are part of a fragile multiprotein 
 complex , the in situ covalent capture prevents potential 
 loss of the targets after cell lysis . because photoaffinity probes 
 are generally used in excess relative to their targets in order to 
 drive the formation of the target  drug complexes , nonspecific 
 targets can also be captured during photo - cross - linking . to overcome 
 this problem ,
two strategies have been employed : ( i ) use ligands with 
 higher affinity so that the photoaffinity probes can be used at lower 
 concentrations ; and ( ii ) use photoaffinity labels that generate reactive 
 intermediates with high , yet selective reactivities 
 toward the ligand - bound targets . to this end , only a few photoaffinity 
 labels have been reported in the past 40 years ( chart 1 ) , including phenyl azide , diazirine ( da ) , and benzophenone 
 ( bp ) .
while these photoaffinity labels 
 have shown tremendous versatility in biomedical research , nevertheless 
 they have two major shortcomings : ( i ) 
 the photogenerated nitrene , carbene , and diradical intermediates exhibit 
 extremely short half - lives , leading to very low target capturing yields , 
 and ( ii ) the nitrone , carbene , and diradical intermediates are prone 
 to react nonselectively with any proximal c  h / x  h bonds 
 ( x = n , o , s ) , resulting in high background .
to balance reactivity with specificity , 
 we envisioned that alternative 
 photogenerated intermediates may exhibit longer half - lives and greater 
 functional group selectivity .
indeed , hamachi and co - workers have 
 reported elegant ligand - directed chemistries based on the electrophilic 
 tosyl and acyl imidazolyl groups and demonstrated their exquisite specificity in 
 selective protein target labeling in situ
. inspired by this work , we hypothesized that an 
 appropriately functionalized photoreactive tetrazole could serve as a highly selective , electrophilic photoaffinity 
 label for in situ target capture .
here we report the development of 2-aryl-5-carboxytetrazole 
 ( act , chart 1 ) as a 
 robust photoaffinity label for identification of the targets and off - targets 
 of dasatinib and jq-1two drugs profiled extensively in the 
 literature . compared with da and bp , act 
 gave higher yields in the ligand - directed photo - cross - linking reactions 
 with the recombinant target proteins .
in addition , the act - based probes 
 facilitated the in situ target identification in 
 a manner similar to the da - based ones .
whereas the tosyl and acyl imidazolyl groups were 
 successfully employed in labeling endogenous targets in living cells , 
 they are not ideal affinity labels for general target identification 
 because of concerns about their stability in cellular milieu .
we hypothesized that act should serve as an 
 ideal photoaffinity label based on the following considerations : ( i ) 
 5-carboxy - substituted 2-aryltetrazoles are photoactive ; ( ii ) placement of a carboxyl group at the c - position of 2h - tetrazole increases the electrophilicity 
 of the photogenerated carboxy - nitrile imine intermediate ; ( iii ) nucleophilic 
 thiol - addition of 2-mercaptobenzoic acid and 3-mercaptopropionic acid to the 
 base - generated carboxy - nitrile imine was reported in the literature ; 
 and ( iv ) the photogenerated carboxy - nitrile imine intermediate should 
 undergo rapid medium quenching when a proximal nucleophile is not 
 available ( figure s1 ) , minimizing 
 the undesired reactions with nonspecific targets .
thus , two series 
 of photoaffinity probes were prepared ( see supporting information for synthetic schemes ) : one series is based on 
 dasatinib , a potent inhibitor of bcr - abl kinase , the src family kinases , as well as btk ( bruton s 
 tyrosine kinase ) ; and the other is based 
 on jq-1 , a potent inhibitor of the bet family of bromodomain proteins ( figure 1a ) . specifically , three photoaffinity labels , act , da , and 
 bp , were attached to dasatinib or jq-1 via the previously reported 
 linkage sites .
the linker length was varied 
 to adjust the distance of the reactive intermediate from the target 
 binding site ( figure 1a ) .
an alkyne tag was placed on the photoaffinity labels to enable 
 the click chemistry - mediated detection and enrichment of the targets 
 from cell lysates . to determine how the 
 attachment of photoaffinity label affects the inhibitory activity 
 and specificity ,
kinome profiling was carried out for dasatinib - derived 
 probes ( figure 1b and table s1 ) , while the in vitro binding assay was performed for jq-1-derived probes ( figure 1c and table s2 ) .
we found that da probes 2a and 2b retained most of their inhibitory activities while act probes 1a and 1b showed modest reduction ( 220-fold ) , 
 particularly for 1a with the shorter linker . in comparison , 

the bp probes exhibited the largest reduction in inhibitory activity 
 ( 25400-fold ) , presumably due to the positioning of a large , 
 flat aromatic structure in the solvent - exposed hinge region .
for the 
 jq-1 series , almost all the photoaffinity probes demonstrated greater 
 inhibitory activities than jq-1 , indicating that the hydrophobic photoaffinity 
 labels form additional interactions with brd2 - 4 outside the shallow 
 and flat canonical binding pocket . in 
 the cell proliferation assays , the photoaffinity label - linked jq-1 
 probes showed potencies similar to the parent jq-1 against leukemic 
 cell line skm-1 , but reduced activities against breast carcinoma mx-1 
 as well as nonsmall cell lung carcinoma nci - h1299 ( table s2 ) , likely due to the permeability difference of the 
 photoaffinity probes and/or the disparate dependency of these cell 
 lines on brd proteins for proliferation .
dasatinib and jq-1-derived 
 photoaffinity probes containing 2-aryl-5-carboxy - tetrazole 
 ( act ) , diazirine ( da ) , or benzophenone ( bp ) photoaffinity label ( pal ) 
 and their biological activities . ( a ) structures of the photoaffinity 
 probes .
a panel of 82 protein kinases were surveyed in this assay , and inhibition 
 constants , ki , are given in micromolar . 

( c ) plots 
 of the inhibition of brd-2 , -3 , and -4 by jq-1-derived photoaffinity 
 probes .
see table s2 in the supporting information for ki values . to compare the efficiency of these three photoaffinity 
 labels in covalently labeling their targets , we treated recombinant 
 btk and brd4 proteins with appropriate probes , and we detected the 
 photo - cross - linked adducts using in - gel fluorescence analysis after 
 the copper - catalyzed click chemistry with rhodamine - azide .
for btk , all probes showed irradiation and ligand - dependent 
 labeling , with 1a and 3a giving the strongest 
 fluorescence ( figure 2a and figure s2 ) , suggesting other factors , 
 e.g. , click chemistry yield , may also affect the overall labeling 
 efficiency .
these probes also selectively labeled btk in the k562 
 cell lysate spiked with recombinant btk protein ( figure s3 ) . for brd4 protein ,
both act- and da - based probes 
 showed uv light- and ligand - dependent labeling , while bp - based probes 6a/6b exhibited strong background labeling , evidenced 
 by lack of signal attenuation in the presence of jq-1 ( figure 2b ) as well as labeling of bsa 
 which was added to the reaction mixture to prevent nonspecific binding 
 of brd4 to the plastic surface ( figure s4 ) . in the k562 cell lysate spiked with recombinant brd4 , act - based 
 probe 4a showed stronger labeling of brd4 than da - based 
 probe 5a , while bp - based probe 6a showed 
 no labeling ( figure s5 ) , presumably due 
 to its nonspecific associations with many cellular proteins .
evaluating 
 the efficiency and selectivity of photoaffinity - labeling 
 of recombinant proteins by the small - molecule probes .
( a ) evaluating 
 the btk labeling efficiency using in - gel fluorescence ( top panels ) . 
 for reaction setup , 0.5 g of btk ( final concentration 0.1 
 m )
, 0.2 m small - molecule probe , 10 m dasatinib 
 ( for competition only ) in 50 l of pbs were used .
for photoirradiation , 
 a hand - held uv lamp with a wavelength of 302 nm for act ( 5 min ) and 
 bp ( 20 min ) or 365 nm for da ( 10 min ) was used .
( b ) evaluating the 
 brd4 labeling efficiency using in - gel fluorescence ( top panels ) . for 
 reaction setup , 0.4 g of brd4 ( final concentration 0.4 
 m ) , 0.2 m small - molecule probe , 5 g of bsa , and 
 10 m jq-1 ( for competition only ) in 50 l of pbs were 
 used .
the equal loading of proteins was verified by sypro ruby staining 
 of the same gels ( bottom panels ) .
see supporting information for procedures of click chemistry with tamra - azide 
 and polyacrylamide gel electrophoresis .
because the photoaffinity probes with the short linker in 
 general 
 exhibited higher labeling efficiency ( figure 2 ) , we decided to focus on these probes in 
 the following comparison studies . to quantify the photo - cross - linking 
 yield , we incubated recombinant protein targets with appropriate dasatinib 
 or jq-1 probes , subjected the mixture to a brief uv irradiation , and 
 analyzed the mixtures by lc - ms
. gratifyingly , act - based probes 1a and 4a showed robust photo - cross - linking with 
 their targets , reaching 60% cross - linking yield for 1a ( figure 3a ) and 95% 
 cross - linking yield for 4a ( figure 3d ) .
in contrast , da - based probes 2a and 5a gave the desired photo - cross - linked products 
 in much lower yields ( figure 3b , 3e ) .
the control experiment showed 
 that the photoactivation efficiencies are similar between the act 
 and da probes ( figure s6 ) .
surprisingly , 
 bp - based probes 3a and 6a did not yield 
 any detectable photo - cross - linked adducts ; instead , the recombinant 
 target proteins showed significant broadening of their mass peaks , 
 suggesting the initial photoadducts , if they are formed , may have 
 undergone fragmentation to generate less than expected lower - molecular 
 weight adducts ( figure 3c , 3f ) .
an alternative explanation is that 
 the benzophenone serves as a photosensitizer to cause nonspecific 
 oxidative damage to the proteins .
importantly , 
 the act - mediated photo - cross - linking with the target protein is ligand - dependent , 
 as addition of dasatinib or jq-1 into the reaction mixture abolished 
 the photoadducts ( figure s7 ) .
in addition , 
 the photo - cross - linking yield showed probe - concentration dependency 
 as increasing amount of act - probe 4a used in the reaction 
 led to a higher photo - cross - linking yield ( figure s8 ) . quantifying the cross - linking efficiency of the photoaffinity labels 
 with recombinant target proteins by lc - ms .
( a  c ) deconvoluted 
 masses of the product mixture after incubating 2.5 m human 
 btk387659 with 25 m dasatinib probe 1a , 2a , or 3a in 100 l of 
 pbs for 15 min followed by photoirradiation with a hand - held uv lamp 
 for 5 min ( 302 nm for act and bp , 365 nm for da ) on ice .
( d  f ) 
 deconvoluted masses of the product mixture after incubating 2.5 m 
 brd444168 with 5 m jq-1 probe 4a , 5a , or 6a in 100 l of pbs followed 
 by photoirradiation with a hand - held uv lamp for 5 min ( 302 nm for 
 act and bp , 365 nm for da ) on ice .
the cross - linking yield was calculated 
 using the following equation : yield% = iphotoadduct/(itarget  protein + iphotoadduct ) , where itarget  protein and iphotoadduct represent the ion counts 
 of the target protein and the photoadduct , respectively , and marked 
 at the upper - right of the spectra . to identify cross - linking sites on the target 
 protein , we digested the probe 1a - treated recombinant 
 btk protein with trypsin , and analyzed the product mixture by lc - ms / ms . 

a tripeptide fragment corresponding to btk488490 with the carboxy - nitrile imine linked with the glu-488 side chain 
 was identified ( figure 4a ) .
it is noted that recombinant btk387659 protein 
 contains 25 glu and 14 asp residues and only glu-488 was detected 
 as labeled , indicating that the photo - cross - linking is ligand - dependent . 

this ligand - directed proximity - driven reactivity is consistent with 
 the probe docking model ( figure 4b ) in which 
 the binding of probe 1a to the kinase active site brings 
 the c of the act ca . 

6.9  away from the carboxylate of glu-488 ; indeed , it is the 
 only nucleophilic side chain within 9.0  from the electrophilic 
 site . certainly , because the act is completely solvent exposed and 
 highly mobile , these distances may vary as the act orients itself 
 dynamically relative to the btk protein .
we propose that the photoadduct 
 is formed via nucleophilic addition of the glu-488 carboxylate to 
 the carboxy - nitrile imine intermediate followed by a 1,4-acyl shift 
 ( figure 4c ) .
this mechanism 
 is consistent with a literature report in which quenching of the in situ generated diaryl nitrile imine by an excess carboxylic 
 acid produced the n-acyl - n-aryl - benzohydrazide product in good yield .
it is conceivable that other nucleophiles such as cys ( figure s9 ) , if they 
 are in close proximity , may also participate in the cross - linking 
 reactions with act for other targets . since a recent report suggested that the photoreactivity of diaryltetrazole 
 can be harnessed for photo - cross - linking with target proteins through 
 their acidic side chains , we compared the intrinsic reactivity of 
 the carboxy - nitrile imine to that of the diaryl - nitrile imine toward 
 glutamic acid ( 10 mm ) in mixed pbs / acetonitrile ( 1:1 ) solution . in 
 the model study ,
the glutamate - quenching product was clearly detected 
 for the diphenyl - nitrile imine ( figure s10 ) .
in contrast , the carboxy - nitrile imine underwent predominant chloride 
 quenching when a weak nucleophile such as glutamic acid is present 
 in solution ( figure s9 ) , suggesting that 
 the observed photo - cross - linking of 1a with glu-488 of 
 the btk enzyme is not merely the result of elevated local concentration 
 of the glutamate near the in situ generated carboxy - nitrile 
 imine . indeed , because of the rapid chloride quenching of the reactive 
 carboxy - nitrile imine , act should be more suitable as a photoaffinity 
 label than the diaryltetrazoles , as the background cross - linking reactions 
 with the nucleophilic side chains present on protein surfaces would 
 be minimal .
determination of the cross - linking site on btk protein and the 
 proposed ligand - directed cross - linking mechanism .
the ms / ms spectrum for probe 1a - modified tripeptide 
 fragment , emr , is shown with the fragment ions annotated on the structure . 

( b ) a docking model of probe 1a bound to btk ( pdb code : 3k54 ) showing a proximal 
 glu-488 residue located on a loop 6.9  away from the c of the tetrazole ring .
( c ) proposed mechanism of the ligand - dependent 
 nucleophilic addition to the carboxy - nitrile imine followed by the 
 o  n acyl shift to generate the specific photoadduct .
encouraged by high in vitro photo - cross - linking 
 efficiency , we sought to assess the efficiency and selectivity of 
 act as a new photoaffinity label for in situ target 
 identification . for comparison
, we included the da - based probes 2a and 5a , as they exhibited excellent biological 
 activities ( figure 1 ) and moderate photo - cross - linking reactivity ( figures 2 and 3 ) . in brief , 
 suspended k562 cells were treated with 1 m probe 1a , 2a , 4a , or 5a for 5 h before 
 uv irradiation ( 5 min for act probe - treated cells at 302 nm ; 10 min 
 for da probe - treated cells at 365 nm ) .
the cells were lysed , and the 
 lysates were reacted with biotin azide prior to pulldown with the 
 streptavidin agarose beads .
western blot 
 analyses revealed that the dasatinib targets , btk , src , and csk , and 
 the jq-1 target , brd4 , were successfully captured by their respective 
 photoaffinity probes , and pretreating the cells with 50 m parent 
 drug , dasatinib or jq-1 , abolished the capture ( figure s11 ) . in - gel digestion of the streptavidin captured 
 proteins on sds - page gel followed by lc - ms / ms analyses produced lists 
 of potential targets . to ensure that the captured proteins are derived 
 from the ligand - dependent photo - cross - linking , high - confidence targets 
 were compiled based on the following two criteria : ( 1 ) at least two 
 unique peptides were identified in the ms , and ( 2 ) the area under 
 the curve ( auc)a measurement of ms signal intensity and reliabilityfor 
 the parent drug - pretreated sample is not detectable . using these criteria , 

six kinases were identified by probe 1a , five of which 
 also appeared in probe 2a - treated cells , indicating act 
 works similarly to da ( figure 5a , table s3 ) . however , probe 1a failed to identify abl protein , presumably due to a lack 
 of proximal nucleophilic side chains near the kinase active site . 
 for jq-1 targets , probes 4a and 5a
successfully 
 captured the bromodomain proteins brd-2 , -3 , and -4 with minimum off - targets 
 ( figure 5b , table s4 ) , suggesting both act and da are efficient 
 in the in situ target identification .
comparison 
 of our data with other literature - reported ms - based target identification 
 studies revealed that these act- and da - based photoaffinity probes 
 performed exceptionally well ( tables s5s6 ) .
venn diagrams of the identified protein targets by ( a ) dasatinib - derived 
 photoaffinity probes 1a and 2a ; and ( b ) 
 jq-1-derived photoaffinity probes 4a and 5a . taken together
, we show that act 
 can serve as an effective photoaffinity 
 label for target identification both in vitro and 
 in live cells .
compared to the existing photoaffinity labels such 
 as bp and da , the main advantage of act lies in its unique photo - cross - linking 
 mechanism , which in principle should lead to reduced background reactions 
 with nonspecific targets as well as a facile mapping of the ligand - binding 
 site .
structurally , act is comparable in size to bp and the electronically 
 stabilized da derivatives such as trifluoromethylaryl diazirine , and 
 it features a modular design with the carboxy group at the c - position of 2h - tetrazole , providing the conjugation 
 handle for a drug molecule and the aryl group responsible for the 
 photoreactivity . compared to da and bp , act showed higher cross - linking 
 yields with the desired targets in vitro ( figure 3 ) , but it produced 
 similar efficiency in target capture in situ in a 
 two - step cross - linking / capture procedure ( figure s11 ) , suggesting additional optimization of the capture step 
 may be necessary in order to achieve higher overall target capture 
 yield . at present ,
an alkyne tag was appended onto the aryl ring to 
 enable the click chemistry - mediated target capture .
however , alternative 
 chemical moieties that are captured covalently by engineered enzymes , 
 e.g. , the haloalkane moiety for halotag and the benzoguanine moiety for snap tag , will be explored in the future for more efficient target capture . 

because of its unique cross - linking mechanism , a potential drawback 
 of act is that a suitable nucleophile needs to be present near the 
 ligand - binding site for a target to be captured and identified , which 
 may result in false negative ; for example , abl kinase was not identified 
 by 1a in this study . in principle , this limitation can 
 be potentially overcome by increasing the linker length between act 
 and the ligand to allow the survey of a larger area surrounding the 
 ligand - binding pocket . in summary , we have developed a new photoaffinity 
 label , 2-aryl-5-carboxytetrazole 
 ( act ) , for efficient in situ target capture and subsequent 
 identification .
the attachment of act to two drug molecules was generally 
 well tolerated without significantly altering the binding affinity 
 and specificity .
compared with da and bp , act provides a unique mechanism 
 of target capture through which the photogenerated carboxy - nitrile 
 imine intermediate reacts with a proximal nucleophile near the target 
 active site . as a result
, act displayed the cleanest and most efficient 
 cross - linking with the recombinant target proteins in vitro among the three photoaffinity labels tested . in the in situ target identification studies with two previously profiled drugs , 
 dasatinib and jq-1 ,
act successfully captured the desired targets 
 in both cases with an efficiency comparable to da .
while aniline was 
 used as the aryl group in the present study , a wide range of heterocycles 
 will be explored in the future with a goal to identify acts with enhanced 
 solubility and photo - cross - linking reactivity .
one microliter 
 of 0.5 mm dasatinib in 
 dmso ( for competition experiments ) or dmso ( without dasatinib competition ) 
 was added to 0.5 g of btk in 50 l of pbs .
after incubation 
 at r.t . for 15 min , 1 l of 10 m photoaffinity probe 
 in dmso was added . after additional incubation at r.t . for 30 min , 

the mixture was irradiated with a hand - held 302 nm uv lamp , ca .
a premixed 
 click reaction cocktail ( 6 l , 1:3:1:1 of 50 mm cuso4 in water/1.7 mm tbta in 1:4 dmso - buoh/50 
 mm tcep in water/1.25 mm tamra - azide in dmso ) was added , and the reaction 
 mixture was incubated at r.t . for 1 h. after 1 h
, 500 l of 
 cold acetone was added , and the mixture was left at 20 c 
 overnight .
the mixture was then centrifuged at 17,200 g at 4 c for 20 min and the pellet was collected . to the pellet 

was added 30 l of 1  sds sample buffer , and the mixture 
 was boiled at 95 c for 10 min before sds - page with 420% 
 bis - tris gel using mops as running buffer .
one microliter of 0.5 mm dasatinib in 
 dmso ( for competition experiments ) or dmso ( without dasatinib competition ) 
 was added to 0.5 g of btk in 50 l of 2 mg / ml k562 cell 
 lysate in pbs .
after incubation at r.t . for 15 min , 1 l of 
 10 m photoaffinity probe in dmso was added .
the mixture was irradiated with a hand - held 
 302 nm uv lamp , ca .
23 cm from the top of 
 the sample . a premixed click reaction cocktail ( 6 l , 1:3:1:1 
 of 50 mm cuso4 in water/1.7 mm tbta in 1:4 dmso - buoh/50 mm tcep in water/1.25 mm tamra - azide in dmso ) 
 was added , and the reaction mixture was incubated at r.t . for 1 h. 
 after 1 h
, 500 l of cold acetone was added and the mixture 
 was left at 20 c overnight .
the mixture was then centrifuged 
 at 17,200 g at 4 c for 20 min , and the pellet 
 was collected . to the pellet
was added 30 l of 1  sds 
 sample buffer , and the mixture was boiled at 95 c for 10 min 
 before sds - page with 420% bis - tris gel using mops as running 
 buffer .
one microliter of 0.5 mm ( + ) -jq-1 in dmso 
 ( for competition experiments ) or dmso ( without competition ) was added 
 to 0.4 g of brd4 and 5 g of bsa ( added to reduce nonspecific 
 binding to the vial surface ) in 50 l of pbs .
after incubation 
 at r.t . for 15 min , 1 l of 10 m photoaffinity probe 
 in dmso was added .
after additional incubation at r.t . for 30 min , 
 the mixture was irradiated with a hand - held 302 nm uv lamp , ca .
a premixed 
 click reaction cocktail ( 6 l , 1:3:1:1 of 50 mm cuso4 in water/1.7 mm tbta in 1:4 dmso - buoh/50 
 mm tcep in water/1.25 mm tamra - azide in dmso ) was added , and the reaction 
 mixture was incubated at r.t . for 1 h. after 1 h
, 500 l of 
 cold acetone was added and the mixture was left at 20 c 
 overnight .
the mixture was then centrifuged at 17,200 g at 4 c for 20 min and the pellet was collected . to the pellet 

was added 30 l of 1  sds sample buffer , and the mixture 
 was boiled at 95 c for 10 min before sds - page with 420% 
 bis - tris gel using mops as running buffer .
one microliter of 0.5 mm ( + ) -jq-1 in 
 dmso ( for competition experiments ) or dmso ( without competition ) was 
 added to 0.1 g of brd4 in 50 l of 2 mg / ml k562 lysate 
 in pbs .
after incubation at r.t . for 15 min , 1 l of 10 m 
 photoaffinity probe in dmso was added . after additional incubation 
 at r.t . for 30 min ,
the mixture was irradiated with a hand - held 302 
 nm uv lamp , ca .
23 cm from the top of the 
 sample . a premixed click reaction cocktail ( 6 l , 1:3:1:1 of 
 50 mm cuso4 in water/1.7 mm tbta in 1:4 dmso - buoh/50 mm tcep in water/1.25 mm tamra - azide in dmso ) 
 was added , and the reaction mixture was incubated at r.t . for 1 h. 
 after 1 h
, 500 l of cold acetone was added and the mixture 
 was left at 20 c overnight .
the mixture was then centrifuged 
 at 17,200 g at 4 c for 20 min and the pellet 
 was collected . to the pellet
was added 30 l of 1  sds 
 sample buffer , and the mixture was boiled at 95 c for 10 min 
 before sds - page with 420% bis - tris gel using mops as running 
 buffer .
plates were stamped with 5 l 
 of kinase buffer ( life technologies # pr4940d ) containing recombinant 
 kinase ( 2.510 nm final concentration ) , eu or tb labeled antibodies 
 ( his or gst ; 0.52 nm final concentration ) , and fluorescently 
 tagged probe ( 3200 nm final concentration ) .
appropriate probes 
 were diluted in kinase buffer , and 120 l of compound was added 
 to the plate using biomex fx .
example for btk kinase : btk ( invitrogen , pv3363 ) 
 was added to 5 l of kinase buffer ( # pr4940d ) to a final concentration 
 of 10 nm , supplemented with 2 nm tb labeled anti - his antibody and 
 200 nm oregon green labeled probe .
afterward , 120 l of diluted 
 compound in kinase buffer was added and the plate was incubated at 
 r.t . for 2 h.
the plate was read on an envision plate reader , and 
 the ki values were calculated using the 
 assay explorer software .
one hundred million k562 cells were 
 plated in 20 ml of dmem media ( 5 million cells / ml ) without 
 fbs and antibiotics .
twenty l of 50 mm unmodified ligand ( dasatinib 
 or ( + ) -jq-1 ) in dmso ( competition experiments ) or dmso ( without competition , 
 control ) was added to the cells , and the mixture was incubated for 
 30 min ( 37 c , 5% co2 , gentle shaking ) . afterward
, 
 20 l of 1 mm probe in dmso ( all experiments except the control ) 
 or dmso ( control ) was added ( final competitor concentration = 50 m , 
 final probe concentration = 1 m ) , and the sample was kept in 
 the incubator for 5 h ( 37 c , 5% co2 , gentle shaking ) . after 5 h of incubation
, 
 cells were washed twice with 2 ml of pbs and then resuspended in 2 
 ml of pbs in 35 mm petri dishes .
the mixture was irradiated with a 
 hand - held 302 nm uv lamp , ca .
pbs was changed to 2 ml of 0.02% 
 tween-20 in pbs , and a protease inhibitor cocktail was added ( amresco , 
 # m250 ) .
the suspended cells were lysed with sonication ( 10  
 10 s with 10 s breaks , 40% power ) on ice .
the lysate was centrifuged 
 ( 20 min , 17,200 g , 4 c ) and filtered through 
 a 0.2 m membrane .
the protein concentration was measured to 
 be 812 mg / ml using the bca assay .
click reaction 
 was performed by following a published procedure . in brief , 10 mg of cell lysate was diluted with pbs to 
 5 ml to obtain a final concentration of 2 mg / ml . to the above solution , 
 113 l of 5 mm azide - peg3-biotin ( aldrich , # 762024 ) 
 in dmso , 113 l of 50 mm tcep in pbs , 340 l of 1.7 mm 
 tbta in 1:4 dmso - buoh , and 113 l 
 of 50 mm cuso4 were added .
the mixture was gently mixed 
 at r.t . for 1 h before 45 ml of acetone
after centrifugation , the 
 protein pellet was collected , washed with 2  10 ml of cold methanol , 
 and redissolved in 14 ml of 0.1% sds in pbs .
prewashed streptavidin 
 agarose beads ( 60 l , thermo scientific , # 20347 ) were added , 
 and the mixture was rocked at 4 c overnight . the beads were 
 washed with 3  1 ml of 0.1% sds in pbs followed by 5  
 1 ml of pbs . then , 60 l of 2  sds sample buffer was added 
 and the mixture was boiled at 95 c for 12 min before samples 
 were loaded onto sds - page gel .
rplc - ms was performed using 
 an agilent 1100 hplc coupled to an agilent 
 lc / msd tof running masshunter workstation acquisition b.04.00 .
data 
 was deconvoluted in masshunter qualitative analysis b.07.00 using 
 the maximum entropy algorithm with a 0.5 da mass step , proton mass 
 adduct , and baseline subtract factor 7.0 .
the site 
 of modification of btk by probe 1a was determined by 
 in - gel trypsin digestion of the band corresponding to the protein 
 after labeling with 1a as described in the following 
 reference : shevchenko , a. evaluation of the efficiency of in - gel digestion 
 of proteins by peptide isotopic labeling and maldi mass spectrometry .
lc - ms / ms analysis was performed using a waters 
 nanoacquity hplc system coupled to a thermo fisher scientific fusion 
 mass spectrometer .
separation of the peptides was achieved using a 
 thermo easyspray pepmap column ( es802 ; c18 , 2 m , 100  , 
 75 m  25 cm ) at a flow of 0.25 l / min , with a gradient 
 starting at 5% b ( b = 0.1% formic acid in acetonitrile , a = 0.1% formic 
 acid in water ) , ramping to 15% b at 2 min , 1535% b over 20 
 min , followed by a 5 min ramp to 80% b , washing for 6 min at elevated 
 flow ( 0.4 l / min ) , before returning to the starting conditions . 

the fusion source was operated at 1.9 kv in positive ion mode with 
 ms detection in the orbitrap using 120 k resolution .
the modified 
 tripeptide was identified by its fragmentation spectrum that resulted 
 from quadrupole isolation of the triply charged ion using an isolation 
 window of 1.8 m / z , and fragmentation 
 via hcd at 26% collision energy , with fragment ion detection in the 
 orbitrap at 15k resolution .
protein from in situ enriched 
 samples was eluted from beads with 100 l of 2  
 lds - page sample buffer ( invitrogen ; 141 mm tris base , 106 mm trishcl , 
 2% lds , 10% glycerol , 0.51 mm edta , 0.22 mm serva blue g , 0.175 mm 
 phenol red , ph 8.5 ) , and the mixture was heated to 80 c for 
 10 min .
a 20-l sample was applied to sds - page running with 
 412% bis - tris gel and mops running buffer to remove the detergent . 
 in - gel digestion
five microliters , representing 10% of each sample , 
 was loaded via waters nanoacquity autosampler onto an acclaim pep 
 map precolumn ( p / n 164535 ) with online trapping and salt removal ( trapping 
 flow rate at 5 l / min for 3.5 min ) .
analytical separation was 
 performed over a 90 min run using an easy spray column ( es802 ) heated 
 to 45 c .
reverse phase gradient was delivered at a flow rate 
 of 0.225 l / min by waters nanoacquity hplc as follows : 0 min 
 10% b , 55 min 25% b , 60 min 40% b , 60.1 min 98.0% b , 65.1 min 10% 
 b , 89.0% b , where b is 0.1% formic acid in acetonitrile .
spectra were 
 collected on a thermo fisher scientific fusion mass spectrometer using 
 the following parameters : 2.1 kv spray voltage , 275 c transfer 
 tube temperature , 3501500 m / z scan range with a quadrupole isolation window of 1.6 m / z , ms1 in the orbitrap at 120 k resolution , ms2 
 by cid in the ion trap with rapid speed , ms2 scans collected with 
 top speed 3 s cycle , dynamic exclusion with repeat count 1 if occurs 
 within 30 s and exclude for 60 s. mips on with charge states 27 
 allowed with 4e5 agc orbitrap and 2e3 ion trap settings .
raw files were processed 
 by proteome discoverer ( v 2.1.081 ) and searched by mascot ( v 2.4.0 ) 
 using the uniprot human database ( downloaded 08 - 10 - 2015 ) . ms1
tolerance 
 was set to 20 ppm , and ms2 tolerances were set to 0.8 da .
label - free 
 quantitation was performed with the precursor ions area detector function . 

areas under the curve ( aucs ) less than 2.0e5 were determined below 
 loq based on previous studies on the performance of the instrument 
 using proteomic reagent standards .
thresholds per experiment were 
 set for significant differences dependent upon the determination of 
 potential sample loading bias by comparing total ion chromatogram 
 ( tic ) intensity between paired injections (  competition ) and 
 average signal from nonspecifically binding protein background .
 photoaffinity labels are powerful 
 tools for dissecting ligand  protein 
 interactions , and they have a broad utility in medicinal chemistry 
 and drug discovery . 
 traditional photoaffinity labels work through 
 nonspecific c  h / x  h bond insertion reactions with the 
 protein of interest by the highly reactive photogenerated intermediate . 
 
 herein , we report a new photoaffinity label , 2-aryl-5-carboxytetrazole 
 ( act ) , that interacts with the target protein via a unique mechanism 
 in which the photogenerated carboxynitrile imine reacts with a proximal 
 nucleophile near the target active site . in two distinct case studies , 
 
 we demonstrate that the attachment of act to a ligand does not significantly 
 alter the binding affinity and specificity of the parent drug . 
 compared 
 with diazirine and benzophenone , two commonly used photoaffinity labels , 
 in two case studies act showed higher photo - cross - linking yields toward 
 their protein targets in vitro based on mass spectrometry 
 analysis . in the in situ target identification studies , 
 
 act successfully captured the desired targets with an efficiency comparable 
 to the diazirine . 
 we expect that further development of this class 
 of photoaffinity labels will lead to a broad range of applications 
 across target identification , and validation and elucidation of the 
 binding site in drug discovery . 
Introduction
Results and Discussion
Experimental Section