lysine acetylation 
 plays a critical role in the regulation of transcription , 
 metabolism , and other central biological functions .
acetylation of 
 lysine residues can impact protein and genome function through multiple 
 mechanisms , including physical relaxation of histone  dna interactions , recruitment of bromodomain - containing effector 
 proteins , covalent active - site modification , and regulation of protein stability .
lysine acetylation is a dynamic ptm that represents 
 an equilibrium between the activity of two opposing enzyme classes : 
 lysine acetyltransferase ( kat ) enzymes , which impose the mark , and 
 lysine deacetylases ( kdacs ) , which remove it .
while kdacs have been extensively investigated as epigenetic drug 
 targets , several analyses indicate kats can also drive cellular transformation 
 and cancer progression in a tissue - specific manner . for example
, fusion 
 of the kat enzyme moz to tif2 is the primary genetic lesion associated 
 with a subset of leukemias and imbues differentiated red blood cells 
 with cancer stem cell - like properties .
nonmutant kat activities can also support oncogenic gene expression 
 programs , functioning as essential coactivators for transcription 
 factors such as c - myc and e2a - pbx gene fusions in cancer .
proteome - wide studies have revealed lysine 
 acetylation is a prevalent 
 ptm , rivaling phosphorylation in terms of substrate diversity with 
 4700 human acetylation sites identified to date .
however , in contrast to the hundreds of known protein kinases , a 
 recent phylogenetic analysis identified only 18 kats in the human 
 genome .
the majority of these canonical 
 kats fall into four families : gcn5/pcaf , p300/cbp , myst , and ncoa , 
 with the rest consisting of transcription factor - related and orphan 
 ( sequence disparate ) kat activities ( supplementary 
 figure s1 ) .
kat family members demonstrate significant intrafamily 
 but little interfamily sequence homology , hindering bioinformatics 
 approaches to kat discovery and classification .
the functional characterization 
 of kats is also limited by their regulation by protein partners and 
 ptms , factors that are difficult to recapitulate in vitro .
furthermore , while individual kats have been shown to be susceptible 
 to inhibition by small molecules and cellular acetyl - coa / coa ratio , 
 methods for comparing the selectivity of these perturbations among 
 multiple kats in parallel do not exist .
thus , our 
 ability to discover and characterize acetylation - mediated signaling 
 would be greatly advanced by the development of new methods for the 
 global analysis of kat activity in cellular contexts .
chemoproteomic 
 profiling provides a powerful alternative to traditional 
 biochemical assays for measuring enzyme activity in complex biological 
 settings . in this approach , also commonly referred to as activity - based 
 protein profiling ( abpp ) , active - site probes for an enzyme class of 
 interest are modified with chemical handles enabling detection or 
 affinity enrichment .
covalent labeling or enrichment of an enzyme 
 by the affinity probe is then used as a proxy for enzyme activity .
chemoproteomic probes for kdac enzymes have 
 been used to discover novel kdac complexes and characterize inhibitor selectivity in cell lysates .
similar approaches have also been 
 pursued to study kat enzymes , utilizing electrophile - containing analogues 
 of the coa cofactor .
however , these probes have not 
 been widely applied to profile kat activity , owing to the fact that 
 most kats do not utilize mechanisms involving active - site nucleophiles . here
we report a general strategy for 
 chemoproteomic profiling 
 of kat activity ( figure 1a ) .
bisubstrate inhibitors 
 targeting three phylogenetically distinct kat families were converted 
 to clickable photoaffinity probes to enable kat labeling and detection . 

cofactor - based affinity probes quantitatively report on kat - inhibitor 
 interactions , are applied to identify a previously unknown acyltransferase 
 activity possessed by the canonical kat enzyme gcn5 , and report on 
 kat activity in cell lysates .
ms / ms 
 profiling of probe targets led to the identification of two noncanonical 
 kat enzymes , highlighting the existence of several orphan lysine acetyltransferases 
 present in the human genome .
in addition to providing insight into 
 the global selectivity and sensitivity of coa - based chemical proteomic 
 probes that will guide future applications , these studies demonstrate 
 the ability of chemical tools for profiling kat activity to provide 
 new insights into kats and their molecular interactions in complex 
 biological contexts .
we envisioned a 
 general strategy for chemical profiling of kat 
 activity based on combining molecular recognition elements from kat 
 bisubstrate inhibitors with a clickable photoaffinity tag for covalent 
 cross - linking and detection ( figure 1a ) .
pioneered 
 by cole and co - workers , kat bisubstrate inhibitors link coa with the 
 -amino group of a lysine - containing peptide to form high affinity 
 interactions with both the substrate and cofactor binding sites of 
 kat enzymes .
these molecules inhibit 
 kat activity with nanomolar potencies , with selectivity for specific 
 kats encoded by the sequence of the bisubstrate peptide . on the basis 
 of literature precedent
, we hypothesized that modification of the 
 n - termini of kat bisubstrate inhibitors might be tolerated without 
 a large loss in inhibitory potency .
this provides a potential site 
 for incorporation of the clickable photoaffinity element benzophenone - l - propargylglycine ( bpyne ; figure 1 ) , 
 necessary for covalent labeling and detection . to test the scope of this approach , we synthesized a suite of kat 
 probes based on bisubstrate scaffolds that have been shown to target 
 three major families of kats : p300/cbp ( lys - coa - bpyne ; 1 ) , gcn5/pcaf ( h3k14-coa - bpyne ; 2 ) , and myst ( h4k16-coa - bpyne ; 3 ) ( figure 1 ; supplementary 
 scheme s1 ) . kat probes 13 were constructed from bpyne - peptide - bromoacetamide precursors , 
 synthesized on rink amide resin utilizing an orthogonal lys - dde protecting 
 group and postcleavage hplc purification .
nucleophilic displacement 
 of bpyne - peptidyl - bromoacetamides , with commerical coa , followed by 
 final hplc purification provided probes 13 on scales ( 1100 mol ) sufficient for biological 
 evaluation ( supplementary scheme s1 ) . to test the affect of our structural modifications on molecular recognition 
 of kat enzymes , we assayed probes 13 against recombinant p300 , pcaf , and mof ( myst1 ) and compared their 
 inhibitory activity to that of non - bpyne - containing  parent  
 inhibitor scaffolds ( 46 ; supplementary figure s2 ) .
assayed at a single 
 concentration ( 1 m ) , probes 13 demonstrate inhibitory potencies and selectivities that closely 
 mimic those of parent inhibitor scaffolds 46 ( supplementary figures s3 and s4 ) .
dose - response analysis of lys - coa - bpyne ( 1 ) demonstrated 
 an ic50 of 26.7 nm toward p300 ( 95% confidence interval 
 [ ci95 ] = 11.7560.64 ) , within error of the inhibition 
 by parent compound 4 ( ic50 = 34.5 nm , ci95 = 17.567.8 nm ; supplementary 
 figure s5 ) .
similar results are seen upon comparison of h3k14-coa - bpyne 2 and parent bisubstrate 5 .
together , these results 
 suggest the bpyne subunit has minimal effects on kat active - site recognition .
next , we 
 evaluated the utility of 13 as kat 
 labeling reagents in vitro .
kat probes 13 were incubated with purified recombinant kats , photoirradiated 
 at 365 nm , and subjected to cu - catalyzed [ 3 + 2 ] cycloaddition (  click 
 chemistry  ) with a fluorescent azide tag . sds - page analysis of labeling reactions demonstrated dose - dependent 
 fluorescent labeling of kats at probe concentrations between 1 and 
 10 m .
notably , each probe showed sensitive detection of the 
 kat enzyme family it was designed to target , i.e. , kat probe 1 reacted effectively with p300 ( p300/cbp ) , probe 2 with pcaf ( gcn5/pcaf ) , and probe 3 with mof ( myst ) ( figure 2a ) .
fluorescent labeling required probe , photo - cross - linking , 
 and cu ; omission of any single component abolished labeling 
 ( supplementary figure s6 ) . kat probes 2 and 3 , which are 3 kda , caused a significant 
 gel shift upon photoirradiation that was visible upon coomassie staining 
 ( figure 2a , supplementary 
 figure s6 ) .
this property allowed us to quantify and optimize 
 our photo - cross - linking conditions using gel densitometry . in our 
 optimized photo - cross - linking protocol
, we found that 3340% 
 of pcaf was covalently labeled by 2 after 60 min of irradiation 
 at 365 nm on ice .
these conditions are consistent with the literature , and cross - linking yields were not found to increase 
 with extended photoirradiation times .
( c ) relative labeling of pcaf by clickable ( left ) and fluorescent 
 ( right ) photoaffinity probes at low ( 0.1 m ) and high ( 1 m ) 
 probe concentrations .
the alkyne handle of probes 13 was chosen for its minimal footprint and versatility toward conjugation 
 of diverse azide reporters .
however , it is not strictly necessary 
 for ex vivo affinity profiling applications . in order to evaluate 
 the utility of our bioorthogonal detection strategy
, we compared the 
 labeling of pcaf by clickable probe 2 with a fluorescent 
 kat photoaffinity probe , tamra - h3k14-coa 7 ( figure 2b ) . both probes
facilitate fluorescent detection 
 of pcaf at high probe concentrations ( 1 m ) , suggesting fluorescent 
 bisubstrates may be useful profiling agents for kat enzymes .
however , 
 clickable probe 2 exhibits visibly greater labeling than 
 fluorescent probe 7 at low probe concentrations ( 0.1 
 m ; figure 2b ) .
this suggests click chemistry 
 detection strategies improve probe sensitivity , possibly by abrogating 
 negative interactions of the fluorescent tamra reporter on probe - protein 
 recognition that reduce photo - cross - linking .
moving toward more complex settings , we assessed the specificity 
 and selectivity of each kat probe ( 13 ) using a cocktail composed of p300 , pcaf , and the myst family acetyltransferase 
 mof .
specific probe labeling events were defined as those susceptible 
 to competition by 50100 equiv of parent kat bisubstrate inhibitors 46 ( supplementary 
 figure s7 ) , while selectivity refers to the subset of kat enzymes 
 labeled by each probe .
interestingly , each probe showed specific labeling 
 of a unique subset of kat enzymes at 1 m , even in the presence 
 of other kat superfamily members .
for example , lys - coa - bpyne 1 showed strong labeling of p300 and pcaf but did not significantly 
 label the myst family member mof ( figure 3 ) . 

the p300 signal was specifically competed in the presence of excess 
 parent inhibitor , while a small portion of the pcaf signal remained , 
 suggesting a combination of specific and nonspecific interactions 
 between the 1-pcaf pair .
h3k14-coa - bpyne 2 demonstrated a clear preference for specific labeling of pcaf and 
 weaker , but detectable , labeling of mof ( figure 3/supplementary figure s8 ) .
h4k16-coa - bpyne 3 most strongly labeled the myst family member mof and exhibited 
 fainter labeling of p300 and pcaf ( figure 3 ) .
in addition to its parent bisubstrate , the mof-3 interaction 
 was competed by excess lys - coa ( 4 ) , which had no similar 
 effect on the interaction of pcaf with 2 ( supplementary figure s8 ) .
for all three probes , 
 selectivities were found to be reduced at higher probe concentrations 
 ( 10 m ) , where 13 exhibit 
 considerable cross - reactivity ( supplementary figure 
 s8 ) .
given its combination of broad - spectrum reactivity and 
 straightforward synthesis of probe and competitor , these findings 
 suggest lys - coa - bpyne 1 may be the most well - suited of 
 the probes for applications requiring a general chemoproteomic reporter 
 of kat activity .
in contrast , when applied at suitably low concentrations , 
 peptidyl - kat probes 2 and 3 may be better 
 suited for applications that require more selective labeling of specific 
 kat families or as components of kat chemoproteomic probe cocktails 
 designed to achieve broad superfamily coverage .
selectivity of kat labeling by probes 13 ( 1 m ) assayed in a mixture of proteins from the p300/cbp , 
 gcn5/pcaf , and myst family .
specific labeling events show sensitivity 
 to competition by parent bisubstrate inhibitors ( 46 ; 200 equiv ) .
having demonstrated the 
 ability of our probes to label three classes of kats , we next sought 
 to investigate their ability to report on changes in kat activity 
 resulting from exposure to diverse molecular stimuli .
first we investigated 
 their ability to report on the affinity and selectivity of small molecule 
 inhibitors .
co - incubation of pcaf with the 
 known kat inhibitor garcinol decreased labeling by kat probe 2 in a dose - dependent manner ( figure 4 ) .
quantification of fluorescent pcaf labeling by gel densitometry 
 yielded an ic50 of 4.5  1.2 m for garcinol , 
 consistent with the literature ic50 value of 5 m .
similarly , labeling of p300 by lys - coa bpyne 1 was sensitive to inhibition by the small molecule inhibitor 
 c646 ( supplementary figure s9 ) .
( a ) fluorescent 
 and coomassie gels from pcaf - garcinol competition experiment . conditions : 2 ( 1 m ) ,
garcinol ( 0 , 0.08 , 0.16 , 0.33 , 0.63 , 1.26 , 
 2.5 , 3.75 , 5 , 10 , 20 , 40 m ) .
( b ) dose - response analysis of 
 competitive labeling generated via gel densitometry analysis of fluorescent 
 labeling by kat probe 2 .
in addition to small molecule inhibition , recent studies 
 have suggested 
 kat activity may be sensitive to changes in cellular acyl - coa pools , 
 providing a potential mechanism to link changes in the metabolic state 
 of the cell to differential histone acylations and epigenetic control 
 of gene expression . to test whether chemoproteomic 
 probes could provide insight into these mechanisms , we investigated 
 the ability of four different acyl - coas ( acetyl , propionyl , butyryl , 
 and crotonyl - coa ) to compete with 2 for the active site 
 occupancy of gcn5 , a kat whose activity has been proposed to be metabolically 
 regulated ( figure 5 ) .
we found that high concentrations of acetyl - coa efficiently competed 
 labeling by probe 2 , consistent with its role as a universal 
 kat cofactor .
propionyl - coa also antagonized labeling , while butyryl - coa 
 was a partial antagonist , and crotonyl - coa did not impede labeling 
 ( figure 5b ) . while the ability of gcn5 to utilize 
 propionyl- and butyrl - coa as cofactors has not been previously explored , 
 our results are consistent with a previous biochemical analysis of 
 human pcaf ( which has a highly homologous kat domain ) .
these studies 
 indicated a kinetic preference for acyl group donors of acetyl - coa 
 ( kcat /
km  
 92 s m )  butyrl - coa 
 and that malonyl - coa was not utilized as a substrate .
indeed , we confirmed gcn5 utilized propionyl- and butyrl - coa 
 as substrates via lc 
conditions : 2 ( 10 m ) , acyl - coas ( 1000 m ) , garcinol ( 40 m ) .
these data suggest competitive 
 affinity profiling provides a useful 
 approach to rapidly gain new insights into kat inhibitor and substrate 
 selectivity .
the activity of kat enzymes such as gcn5 
 and pcaf is ideally studied 
 in cellular settings .
commentary has suggested the failure 
 of conventional high - throughput screening campaigns to yield selective 
 kat inhibitors may be due to the inability of these screens to interrogate 
 the ability of small molecules to disrupt native kats , which can exist 
 as multiprotein complexes . therefore , 
 methods to monitor kat activity directly from cell extracts are potentially 
 valuable for next - generation inhibitor discovery efforts .
overexpression extracts of epitope - labeled atac ( ada two - a containing ) 
 complex constitute an advanced model system applied for the study 
 of gcn5 family kats in their endogenous setting .
we thus asked whether we could use chemoproteomic probe 2 to directly detect kat activity in these systems , without 
 the need for prefractionation , affinity purification , or antibodies . 

accordingly , pcaf overexpression extracts from hek-293 cells were 
 treated with 2 , followed by photo - cross - linking and click 
 chemistry .
weak but detectable labeling of a protein corresponding 
 to the molecular weight of pcaf was observed by fluorescence ( figure 6a ) .
notably , labeling was sensitive to competition 
 by h3k14-coa , and immunoblotting confirmed comigration with pcaf . 
 to further verify labeling , we subjected proteins labeled by 2 in pcaf overexpression extracts to click chemistry with 
 biotin azide , followed by affinity purification , tryptic digest , and 
 lc  ms / ms protein identification .
notably , pcaf peptides were 
 specifically identified in overexpression extracts treated with 2 ( figure 6b ) , but not in extracts 
 pretreated with excess competitor 5 or lysates derived 
 from hek-293 cells transfected with an empty vector control ( figure 6a , bottom , supplementary table 
 s1 ) .
in addition to overexpression extracts , clickable photoaffinity 
 probes 1 and 2 are capable of detecting 
 p300 and pcaf , respectively , when spiked into hela cell proteomes 
 ( supplementary figure s11 ) .
we used this 
 characteristic to assess the sensitivity of probe 2 and 
 estimate a lower limit at which gcn5/pcaf family enzymes may be detected . 

this is especially relevant since our inability to enrich pcaf from 
 empty vector transfected hek-293 lysates indicates expression level 
 may be a limiting factor for kat identification .
we found that probe 2 could detect as low as 2.5 pmols of pcaf in a standard gel - based 
 experiment against a proteomic background ( figure 6b ) .
these findings calibrate the ability of chemoproteomic 
 probes to monitor kat activity in model systems and cell lysates and 
 may be useful for the development of chemoproteomic approaches to 
 screen for selective inhibitors of kats and kat - containing multiprotein 
 complexes .
( b ) proteins identified 
 in lc  ms / ms experiments as targets of h3k14-coa - bpyne 2 in pcaf transfected hek-293 extracts . control = competitor 
 treated lane , ev = extract derived from hek-293 cells transfected 
 with empty vector .
( c ) limit of detection of recombinant pcaf spiked 
 into hela cell proteome .
conditions : 2 ( 1 m ) , 
 proteome ( 7 g ) , recombinant pcaf ( 0 , 6.25 , 5 , 3.75 , 2.5 , 1.25 
 pmol ) . having demonstrated the utility of chemoproteomic 
 probes for the targeted study of kat activity in cellular contexts , 
 we last sought to explore their utility for the discovery of potentially 
 novel kat activities directly from cancer cell proteomes . labeling 
 of whole cell extracts by 13 demonstrated 
 a distinct pattern of specific protein labeling events for each probe , 
 with kat probe 1 targeting the largest number of proteins 
 ( figure 7 ) . to identify the proteomic targets 
 of broad - spectrum kat probe 1
, hela cells were lysed , 
 photo - cross - linked in the presence of 1 , and subjected 
 to click chemistry with biotin - azide followed by streptavidin enrichment , 
 tryptic digest , and lc  ms / ms analysis .
we identified 16 proteins 
 that were abundant ( > 5 spectral counts ) and showed > 5-fold preferential 
 enrichment in the absence of competitor ( figure 7b , supplementary table s2 ) .
peptides matching 
 canonical kat p300 and cbp were not observed in our ms / ms data set , 
 likely due to their low abundance in whole cell lysates ( confirmed 
 by western blot ; supplementary figure s12 ) .
of the 16 proteins , 2 were acetyltransferases ( green ) , 5 were 
 coa - binding proteins related to primary metabolism ( red ) , with the 
 remaining hits composed of highly abundant proteins and members of 
 the proteasome regulatory complex . to validate lc 
ms / ms identifications , 
 we performed probe labeling experiments of two overexpressed and purified 
 targets of 1 : atp - citrate lyase ( acly ) , the highest abundance 
 pulldown target of 1 , and n--acetyltransferase 
 50 ( naa50 ) , an acetyltransferase . both enzymes exhibited competitor - sensitive 
 labeling , indicative of specific molecular recognition by kat probe 1 ( figure 7c ) .
conserved domain analysis 
 indicated that each protein specifically labeled by 1 either contains or is closely associated with a protein containing 
 a binding site for an adenine nucleotide - containing cofactor ( coa , 
 atp , or nad(p ) ; figure 7c ) .
the off - target 
 engagement of adenosine - binding proteins by kat probe 1 parallels the widespread reactivity that has been observed for atp- 
 and gtp - containing chemical proteomic probes in biological settings .
this is likely to be a general limitation of chemical proteomic 
 probes incorporating adenine cofactor - based chemical scaffolds and 
 highlights opportunities for design improvements as well as the requirement 
 for orthogonal validation strategies to support chemical proteomic 
 kat discovery efforts .
cofactor - based affinity profiling of endogenous kat activity 
 in 
 a cancer cell proteome .
( a ) labeling of hela cell proteomes by kat 
 probes 13 ( 10 m ) .
specific 
 labeling events show sensitivity to competition by parent bisubstrates 
 ( 100 equiv ) .
( c ) affinity labeling of recombinant protein 
 verifies acly and naa50 as targets of 1 . in our data
nat10 is a noncanonical kat ( referred to here as orphan kats ) 
 with a gnat - related fold that displays histone and microtubule acetyltransferase 
 activity in cells .
biologically , nat10 has been observed to play a 
 role in the regulation of telomerase function and nuclear shape and 
 was recently identified as a druggable target for the treatment of 
 hutchinson  gilford progeria syndrome .
naa50 ( nat13 ) is the catalytic component of the nate acetyltransferase 
 complex that is required for proper sister chromatid adhesion and 
 chromatin condensation in vivo .
the identification 
 of naa50 as a target of lys - coa - bpyne 1 initially struck 
 us as paradoxical , as this enzyme belongs to the n - terminal acetyltransferase 
 ( nat ) family and has been shown to favor protein acetylation of n - terminal 
 met residues . however , literature investigation 
 revealed that , of the 7 nat catalytic subunits encoded in the human 
 genome , naa50 is the only member to have biochemically characterized 
 -lysine acetyltransferase activity , providing a molecular rationale 
 for its targeting by 1 .
the 
 selective identification of naa50 by 1 suggests that 
 chemoproteomic profiling may have applications in identifying new 
 kat activities present in acetyltransferase families that are distinct 
 in sequence from canonical kats .
the proteomic identification 
 of two orphan kat activities by affinity 
 probe 1 led us to re - evaluate the lysine acetyltransferase 
 literature and consider whether other kat activities were also missing 
 from the list of 18 canonical human kats ( supplementary 
 figure s1 ) .
this analysis identified 14 proteins , including 
 nat10 and naa50 , not in the list of 18 canonical kats for which evidence 
 of lysine acetyltransferase activity has been observed .
sequence alignment 
 and similarity analyses were used to construct an expanded phylogenetic 
 tree of acetyltransferase proteins , divided into canonical ( p300/cbp , 
 gcn5/pcaf , myst , ncoa ) and orphan kats ( figure 8) .
notably , many kats from the initial list of 18 ( atat1 , tf3c4 , 
 elp3 ; supplementary figure s1 ) cluster 
 more closely with orphan kats , indicative of greater sequence similarity . 
 together , these proteins encompass the most comprehensive list of 
 human kats assembled to date .
however , we take care to point out that 
 the experimental evidence supporting the activity of all 32 kats , 
 canonical and orphan , ranges widely . in particular
, this list contains 
 two proteins ( oga / ncoat , an orphan kat , and src1 , a canonical kat ) 
 for which conflicting observations of kat activity have been made .
these discrepancies support the need for universal methodologies 
 capable of directly assaying kat activity in endogenous cells , toward 
 which our current study provides an initial step . in the meantime
, 
 our chemoproteomics - inspired census of kat activity provides fertile 
 ground for functional investigation of orphan kat enzymes using traditional 
 structural and biochemical approaches , studies that may facilitate 
 a more complete understanding of lysine acetylation in living systems . expanded 
 phylogenetic tree of kat enzymes , including canonical 
 kats and orphan kat activities observed in this study or annotated 
 in the literature .
uniprot accession numbers and literature references for kat activity 
 are provided in the supporting information ( table s3 ) .
in 
 summary , here we have described a suite of probes for cofactor - based 
 affinity profiling of kat activity .
we have defined key structural 
 features necessary for the covalent labeling and detection of three 
 families of kat enzymes and demonstrated the utility of these probes 
 to monitor kat activity in settings ranging from purified enzymes 
 to kat overexpression extracts to native proteomes .
chemoproteomic 
 probes of kat activity provided new insights into kat inhibitor and 
 cofactor selectivity and highlighted the existence of several noncanonical 
 orphan kat activities that may contribute to cellular acetylation 
 signaling pathways .
in addition to these advances , it is also important 
 to call attention to the limitations of our initial study .
for example , 
 while we demonstrated the ability of kat affinity probes to report 
 on pcaf activity in hek-293 overexpression extracts , we were unable 
 to detect canonical kat enzymes such as cbp and gcn5 directly from 
 hela proteomes .
this may be due to the low abundance of these kats 
 in whole cell lysates ( supplementary figure s13 ) or the low cross - linking yields of our clickable photoaffinity 
 probes , which covalently label only 3340% of recombinant 
 pcaf in vitro .
this challenge may be addressed in future studies through 
 scale - up , nuclear prefractionation , or utilization of multidimensional 
 protein identification technology ( mudpit ) to increase lc  ms / ms 
 detection sensitivity .
alternatively , 
 chemical proteomic studies of kinase and kdac activity have shown 
 that noncovalent affinity probe resins can enable enrichment of specific 
 enzyme classes without the need for photo - cross - linking , providing another potential route for the analysis of low abundance 
 kats . furthermore , while the activity of kat complexes have been shown 
 to be preserved in cell extracts , these activities would be ideally 
 studied in living cells and probes 13 are not cell - permeable .
cell - penetrating peptides have been used 
 to promote uptake of kat bisubstrate inhibitors , and similar approaches 
 may facilitate live cell profiling of kat activity .
these improvements will be important to expand the scope 
 of chemical proteomic analyses of kat activity .
regardless , the ability 
 of our current suite of chemoproteomic probes to highlight an expanded 
 landscape of catalytic lysine acetylation provides an example of the 
 power of this approach as currently constituted and sets the stage 
 for the development of chemoproteomic strategies to identify kat inhibitors 
 and the functional characterization of canonical and orphan kat activities 
 in cellular settings .
recombinant pcaf , catalytic 
 domain ( aa 492658 ) was obtained from cayman chemical .
recombinant 
 p300 , catalytic domain ( aa 12841673 ) and recombinant gcn5 , 
 catalytic domain ( 497663 ) , were obtained from enzo .
the plasmid 
 encoding recombinant mof , catalytic domain ( aa 147449 ) was 
 obtained from addgene .
his - tagged recombinant mof was expressed in e. coli bl21 and purified via immobilized nickel affinity 
 chromatography using standard conditions .
sds - page was performed using bis - tris nupage gels ( 412% ) 
 and mes running buffer in xcell surelock minicells ( invitrogen ) according 
 to the manufacturer s instructions .
sds - page fluorescence was 
 visualized using an imagequant las4010 digitial imaging system ( ge 
 healthcare ) .
total protein content on sds - page gels was visualized 
 by blue - silver coomassie stain , made according to the published procedure .
separation - based assays for kat activity were 
 performed on a labchip ez reader instrument ( perkinelmer ) kindly provided 
 by dr .
fluorescence assays for the kat enzyme mof 
 were analyzed on a biotek synergy 2 ( biotek ) .
recombinant kat activity was 
 measured by electrophoretic mobility shift assay ( emsa ) as previously 
 reported .
this assay measures the separation 
 of fitc - labeled kat substrate peptides ( histone h3 5 - 23 qtarkstggkaprkqlatk - ahx - fitc ; 
 histone h4 1 - 19 sgrgkggkglgkggakrhr - ahx - fitc ) 
 from their acetylated products following incubation with recombinant 
 kat and acetyl - coa .
p300 and pcaf assays were performed 
 in 30 l of reaction buffer ( 50 mm hepes , ph 7.5 , 50 mm nacl , 
 2 mm edta , 0.05% tween 20 , 10 g / ml bsa ) with kat ( p300 [ 50 
 nm ] or pcaf [ 10 nm ] ) and fitc - peptide ( fitc - h4 for p300 ; fitc - h3 for 
 pcaf ; 1 m ) .
acetylation of fitc - h3/h4 peptide by p300 and pcaf 
 was confirmed by lc 
reactions were plated in 384-well plates , 
 allowed to equilibrate at room temperature for 10 min , and initiated 
 by addition of acetyl - coa ( final concentration = 5 m ) .
plates 
 were then transferred to a lab - chip ez - reader at ambient temperature 
 and analyzed by microfluidic electrophoresis .
optimized separation 
 conditions were downstream voltage of 400 v , upstream voltage 
 of 2900 v , and a pressure of 2.0 psi for fitc - h3 and 
 downstream voltage of 500 v , upstream voltage of 1500 
 v and a pressure of 2.0 psi for fitc - h4 .
percent conversion 
 is calculated by ratiometric measurement of substrate / product peak 
 heights .
percent activity represents the percent conversion of kat 
 reactions treated with inhibitors 16 relative to untreated control kat reactions , measured in triplicate , 
 and corrected for nonenzymatic acetylation .
mof showed low activity 
 toward fitc - h3/h4 peptide substrates and was monitored by fluorogenic 
 kat assay using an unlabeled h4 substrate peptide as previously reported .
dose - response analysis of p300 and pcaf inhibition 
 by kat probes 1 and 2 and parent inhibitors 4 and 5 were performed in triplicate and analyzed 
 by nonlinear least - squares regression fit to y = 
 100/(1 + 10(log ic50 
purified kat enzymes ( 0.55 
 g ) or whole cell proteomes 
 ( 20 g ) were incubated with kat probes 13 ( 1 m probe for recombinant labelings ; 10 m 
 probe for proteomic labelings ) in pbs ( ph 7.0 ) for 1 h. control experiments 
 to correct for nonspecific cross - linking were treated with 13 in the presence of 100 equiv of competitors 46 .
following equilibration , samples 
 were photo - cross - linked on ice for 1 h using a 365 nm uv light in 
 a fb - uvxl-1000 uv cross - linker .
probe labeling was detected by cu(i)-catalyzed 
 [ 3 + 2 ] cycloaddition (  click chemistry  ) .
click reactions 
 were initiated by sequential addition of tamra - azide 8 ( 100 m ; 5 mm stock solution in dmso , structure given in supplementary figure s15 ) , tcep ( 1 mm ; 100 mm 
 stock in h2o ) , tris(benzyltriazolylmethyl)amine ligand 
 ( tbta ; 100 m ; 1.7 mm stock in dmso / tert - butanol 
 1:4 ) , and cuso4 ( 1 mm ; 50 mm stock in h2o ) . 

samples were vortexed and incubated at room temperature for 1 h. cycloaddition 
 reactions were quenched by addition of 5x sds - loading buffer ( strongly 
 reducing ) and subjected to sds - page ( 22 l per well ) .
excess 
 probe fluorescence was removed by destaining in a solution of 50% 
 meoh/40% h2o/10% acoh overnight .
gels were then washed 
 with water and fluorescently visualized using a imagequant las4010 
 ( ge healthcare ) with green led excitation ( max 520550 
 nm ) and a 575df20 filter . for kat probes 2 and 3 , a characteristic intense low molecular weight fluorescence 
 signal 3 kda
was observed ( corresponding to the fluorescently 
 labeled kat probe [ 2 , 3 ] ) , indicative of 
 a high - yielding click chemistry reaction .
hela 
 s3 cells ( atcc ; manassas va ) were cultured at 37 c under 5% 
 co2 atmosphere in a culture medium of dmem supplemented 
 with 10% fbs and glutamine .
hek-293 cells were obtained from the nci 
 tumor cell repository . for isolation of whole cell proteomes ,
hela 
 cells were grown to 8090% confluency , washed 3 with 
 ice - cold pbs , scraped , and pelleted by centrifugation ( 1400 g  3 min , 4 c ) .
after removal of pbs cell pellets 
 were stored at 76 c or immediately processed .
cell pellets 
 were resuspended in 12 ml of ice - cold pbs ( 1020  
 10 cells / ml ) and lysed by sonication ( qsonica xl2000 100 
 w sonicator , 3  10 s pulse , 50% power , 60 s between pulses ) . 

lysates were pelleted by centrifugation ( 14,000 g  
 30 min , 4 c ) and quantified on a qubit 2.0 fluorometer using 
 a qubit protein assay kit .
proteomes were diluted to 2 mg / ml and stored 
 in 1 mg aliquots at 76 c until further processing .
sds - page gels were transferred to 
 nitrocellulose membranes ( novex , life technologies ) by electroblotting 
 at 30 v for 1 h using a xcell ii blot module ( novex ) .
membranes were 
 blocked using startingblock ( pbs ) blocking buffer ( thermo scientific ) 
 for 20 min and then incubated overnight at 4 c in a solution 
 containing the primary antibody of interest ( anti - gcn5 , anti - cbp 
 , cell signaling , 1:1000 dilution ) in the above blocking buffer 
 with 0.05% tween 20 .
the membranes were next washed with tbst buffer 
 and incubated with a secondary hrp - conjugated antibody ( anti - rabbit 
 igg , hrp - linked , cell signaling , 1:1000 dilution ) for 1.5 h 
 at room temperature .
the membranes were again washed with tbst , treated 
 with chemiluminescence reagents ( western blot detection system , cell 
 signaling ) for 1 min , and imaged for chemiluminescent signal using 
 an imagequant las4010 digitial imaging system ( ge healthcare ) .
whole 
 cell proteomes were adjusted to a final protein concentration of 1 
 mg / ml and incubated with the indicated probe ( 1 or 2 ; 10 m ) for 1 h. hek-293 enrichments utilized 0.5 
 mg of proteome as starting material , while hela enrichments utilized 
 1 mg of proteome .
control samples to correct for nonspecific cross - linking 
 were preincubated with each probe s cognate competitor ( 4 or 5 ; 100 equiv ) .
following equilibration , 
 samples were split into 5  200 l aliquots and photo - cross - linked 
 on ice for 1 h using a 365 nm uv light in a fb - uvxl-1000 uv cross - linker . 

cross - linked samples were then recombined and subjected to cu(i)-catalyzed 
 [ 3 + 2 ] cycloaddition with tamra biotin - azide 9 ( supplementary figure s15 ) as previously described . 

final concentrations for click reactions were as follows : hela proteome 
 ( 1 mg / ml in pbs ) , probe 1 ( 10 m ) , tamra biotin - azide 
 ( 40 m ) , tcep ( 1 mm ) , tbta ( 100 m ) , tert - butanol ( 4.8% ) , and cuso4 ( 1 mm ) .
samples were vortexed 
 and incubated at room temperature for 1 h. ice - cold 4:1 meoh / chcl3 ( 2.5 ml ) was then added directly to the reaction mixture 
 and mixed vigorously by vortexing .
the biphasic solution was centrifuged 
 ( 4000 g  20 min , 4 c ) , and protein precipitated 
 at the interface as a solid disk .
liquid layers were carefully discarded , 
 and the resulting precipitate was resuspended in ice - cold 1:1 meoh / chcl3 ( 1 ml ) , sonicated on ice to resuspend , and repelleted by 
 centrifugation ( 14,000 g  10 min , 4 c ) . 

the resulting 
 cell pellet was air - dried to remove excess methanol and redissolved 
 in 1.2% sds ( 1 ml ) using iterative cycles of heating ( 95 c ) 
 and sonication .
redissolved protein was allowed to cool to room temperature 
 and added to 5 ml of pbs to give a final sds concentration of 0.2% . 

samples were then treated with 100 l of streptavidin - agarose 
 resin ( prewashed 3 with 1 ml of pbs ) and rotated for 1 h at 
 room temperature .
streptavidin - agarose bound samples were then washed 
 sequentially with 0.2% sds in pbs ( 3  10 ml ) and pbs ( 3  
 10 ml ) .
samples were then prepared for on - bead digest by reduction 
 with 10 mm tris(2-carboxyethyl)phosphine ( tcep ) and alkylation with 
 12 mm iodoacetamide .
samples were diluted to 2 m urea with 50 mm tris - cl 
 ph 8.0 ( 400 l total volume ) , followed by addition of trypsin 
 and 2 mm cacl2 .
after extraction , tryptic peptide samples were acidified 
 to a final concentration of 5% formic acid and frozen at 80 
 c for lc 
tryptic peptides enriched 
 by probe 1 were loaded onto 
 a reverse phase capillary column and analyzed by lc separation in 
 combination with tandem ms .
peptides were eluted using a gradient 
 of 542% over 40 min with the flow rate through the column 
 set at 0.20 l / min .
data was collected in a dual - pressure linear 
 ion trap mass spectrometer ( thermofisher ltq velospro ) set in a data - dependent 
 acquisition mode .
the 15 most intense molecular ions in the ms scan 
 were sequentially and dynamically selected for subsequent collision - induced 
 dissociation ( cid ) using a normalized collision energy of 35% .
tandem 
 mass spectra were searched against uniprot h. sapiens protein database ( 01 - 13 release ) using sequest ( thermofisher ) .
search 
 parameters were fixed as follows : ( i ) enzyme specificity : trypsin ; 
 ( ii ) variable modification : methionine oxidation and cysteine carbamidomethylation ; 
 ( iii ) precursor mass tolerance 1.40 amu ; and ( iv ) fragment ion 
 mass tolerance 0.5 amu .
only those tryptic peptides with up 
 to two missed cleavage sites meeting a specific sequest scoring criteria 
 ( delta correlation ( cn )  0.08 and charge state dependent 
 cross correlation ( xcorr )  1.9 for [ m + h ] ,  
 2.2 for [ m + 2h ] , and 3.1 for [ m + 3h ] ) were considered as legitimate identifications .
spectral count values 
 depicted in figure 7 represent an average of 
 two biological replicates .
raw spectral counts for biological duplicates 
 of probe - enriched and control experiments are provided in supplementary table s2 .
a pairwise alignment was generated using clustal omega , and a phylogenic tree was constructed using 
 the neighbor - joining method .
all phylogenetic trees were displayed 
 in hyperbolic space using hypertree , with branches of the tree designated 
 by different colors and labeled by name where appropriate .
 lysine acetyltransferases ( kats ) 
 play a critical role in the regulation 
 of gene expression , metabolism , and other key cellular functions . 
 
 one shortcoming of traditional kat assays is their inability to study 
 kat activity in complex settings , a limitation that hinders efforts 
 at kat discovery , characterization , and inhibitor development . to 
 address this challenge , here 
 we describe a suite of cofactor - based 
 affinity probes capable of profiling kat activity in biological contexts . 
 
 conversion of kat bisubstrate inhibitors to clickable photoaffinity 
 probes enables the selective covalent labeling of three phylogenetically 
 distinct families of kat enzymes . 
 cofactor - based affinity probes report 
 on kat activity in cell lysates , where kats exist as multiprotein 
 complexes . 
 chemical affinity purification and unbiased lc  
 ms / ms 
 profiling highlights an expanded landscape of orphan lysine acetyltransferases 
 present in the human genome and provides insight into the global selectivity 
 and sensitivity of coa - based proteomic probes that will guide future 
 applications . 
 chemoproteomic profiling provides a powerful method 
 to study the molecular interactions of kats in native contexts and 
 will aid investigations into the role of kats in cell state and disease . 
Introduction
Results and Discussion
Conclusion
Experimental Details