many biochemical processes in the eukaryotic cell are controlled by the posttranslational modification of proteins with ubiquitin ( ub ) , a 76 amino acid polypeptide that is highly conserved among eukaryotes ( kerscher et  al . , 2006 ) .
ub adopts a globular structure in which all seven lysine residues and the n terminus are solvent exposed and can therefore form linkages with the c terminus of another ub to form poly - ub chains ( ye et  al . , 2012 ) .
the ub linkage type and the chain length encode the fate of a ubiquitinated substrate .
protein modification by k48-linked poly - ub chains is a well - established signal for recognition and initiation of degradation by the 26s proteasome complex , whereas proteins tagged with k63-linked ub chains are known to be involved in nonproteolytic events such as in dna damage responses and immune signaling ( komander and rape , 2012 ) .
the roles of  atypical  poly - ub chains linked through the ub n terminus , k6 , k11 , k27 , k29 , or k33 are less well understood , but proteins tagged in such a way also appear to be directed toward proteasomal degradation ( kulathu and komander , 2012 ; xu et  al . , 2009 ) .
the molecular basis for the biological diversity of poly - ub chains stems from their distinct structural features .
some ub - linkages such as k6 , k11 , and k48 adopt compact structures where adjacent moieties interact with each other whereas linear and k63-linked chains display more open conformations where the linkage site is the only interface , although all of them show a degree of flexibility and dynamics ( ye et  al . , 2012 ) .
this implies that enzymes involved in ubiquitin conjugation ( e1/e2/e3 ligases ) and deconjugation ( deubiquitinating enzymes [ dubs ] ) must bear specific recognition features to create and process different poly - ub chains in their biological context , often involving direct interactions with cognate protein substrates and perhaps with other components of multiprotein complexes they are embedded in ( reyes - turcu et  al . , 2009 ) .
dubs are proteases that mediate the removal or processing of ub chains or linked ubl proteins by hydrolyzing the isopeptide bond between the c - terminal glycine of the distal molecule and the lysine residue in either the proximal ub or the protein substrate .
dubs have recently emerged as key regulators in a multitude of processes from gene transcription to protein degradation and from cell division to cell death ( nijman et  al . , 2005 ; tsou et  al . ,
they consist of a protease family comprising 90 members in human cells that can be grouped into five distinct families including ubiquitin - specific proteases ( usps ) , ubiquitin c - terminal hydrolases ( uchs ) , ovarian tumor domain proteases ( otus ) , and machado - joseph disease ( mjd ) protein family , all of which are cysteine proteases .
available knowledge of dub specificity or mode of regulation in cells is scant due to challenges in isolating dub containing protein complexes or expressing full - length recombinant enzymes ( reyes - turcu et  al . , 2009 ) .
current efforts to study dubs in  vitro generally focus on the production of recombinant enzymes and the use of artificial substrates ( e.g. , ub - amc ) .
such investigations may not always reflect their substrate specificity and selectivity in a cellular environment .
although k48- and k63-linked ubiquitin dimers and oligomers have been accessible through chemoenzymatic synthesis for a  number of years ( pickart and raasi , 2005 ; piotrowski et  al . ,
1997 ) , synthetic access to the less well studied atypical linkages has only been established more recently .
selectively functionalized ub variants have been assembled from peptide building blocks using native chemical ligation - based strategies ( kumar et  al .
, 2010 ; yang et  al . , 2010 ) as well as by optimized solid phase peptide synthesis of full length ub ( el oualid et  al . , 2010 ) . in each of these strategies ,
a mercapto - lysine residue was utilized for ligation with a c - terminal ub thioester followed by desulfurization to obtain an isopeptide linkage containing a native lysine residue . genetic incorporation of protected boc - lysine in escherichia coli followed by orthogonal amine protection schemes and ag(i)-mediated thioester to
the successful genetic incorporation of protected mercapto - lysine derivatives in e.  coli has now also enabled the combination of unnatural amino acid incorporation with the facile ligation methodology enabled by mercapto - lysine derivatives ( virdee et  al . , 2011 ) .
in addition to methods for the generation of native isopeptide - linked ub dimers , there has also been an increased interest in novel straightforward methods for ligation of ub by non - native linkages .
cu(i)-catalyzed triazole formation has been successfully employed for the generation of ub - protein conjugates ( weikart and mootz , 2010 ) as well as ub dimers ( eger  et  al . , 2010 ;
it was further demonstrated that the obtained conjugates are resistant to cleavage by dubs ( weikart and mootz , 2010 ) . as an addition to the repertoire of conventional in  vitro assays , ub - based active site probes , targeting
dub catalytic sites in crude cell extracts were successfully used to discover and profile active enzyme species ( borodovsky et  al .
, 2001 ; de jong et  al . , 2012 ; love et  al . , 2009 ) and small molecule dub inhibitors ( altun et  al . , 2011 ) .
the latter approach does not require biochemical purification of dubs and can report on their activity and inhibition within their biological context , but so far was restricted to single ub or ub linked to a branched peptide as recognition scaffolds ( iphfer et  al . , 2012 ) . in this study
, we have engineered active site probes on a dimeric ubiquitin ( di - ub ) scaffold representing all eight different ub - linkages , using biochemical methods coupled with discovery proteomics and quantitative mass spectrometry to define dub poly - ub linkage specificity in a cellular environment .
activity - based di - ub probes formed of two recombinant ub proteins were designed ( figure  1 ) with the following consideration in mind : the isopeptide bond present in the natural ub dimer was to be replaced by a reactive electrophile for the covalent capture of cysteine protease dubs .
the linkage position was controlled by site - directed mutagenesis and incorporation of the unnatural amino acid azidohomoalanine ( aha ) into the proximal ub using the methionine analog incorporation approach ( kiick et  al . , 2002 ) .
subsequently , the proximal ub was linked by cu(i)-catalyzed 1,4-triazole formation to the c terminus of the distal ub .
molecular modeling indicated similarities in spatial requirements of the linker region between the natural linkage containing a gly to lys isopeptide bond and the reactive electrophilie linked via a 1,4-triazole ( figure  s1 available online ) .
a recent computational study using quantum mechanical / molecular mechanical ( qm / mm ) methods confirmed that k48-linked ub - dimer contains similar geometric features in both native ub dimer and a synthetic triazole - linked ub dimer ( dresselhaus et  al . , 2013 ) .
the comparison was carried out between k48-linked ub dimer with a native isopeptide linkage and a triazole containing dimer based on click conjugation of a k48azphe containing ub . with this approach ,
di - ub probes mimicking linkages to each of  the seven lysine residues or the n - terminal methionine were synthesized .
the ha - tag present on the n - terminal of the distal ub ( derived from ha - ub(1 - 75)-alkyne ) allowed visualization and retrieval for identification using tandem mass spectrometry ( lc - ms / ms ) .
introduction of an alanine residue at position two of ubiquitin ( i2a ) to facilitate n - terminal met / aha cleavage by methionine aminopeptidase ( wang et  al . , 2008 ) was followed by individual k to m point mutations at each of the lysine residues to afford the seven ub mutant plasmids .
the ub mutants and wt ub  were expressed in a methionine auxotrophic e.  coli strain for azidohomoalanine incorporation ( m1aha , k6aha , k11aha , k27aha , k29aha , k33aha , k48aha , k63aha ) and purified ( pickart and raasi , 2005 ) .
maldi - tof ms of the intact proteins displayed efficient n - terminal met / aha cleavage as determined by the strength of signal at 8,504 m / z ( corresponding to the mass of n - terminal cleaved ub ) versus a negligible to minor signal detected at 8,639 m / z ( corresponding to uncleaved ub ) .
lc - ms / ms analysis after digestion with trypsin was also carried out to verify site - specific aha incorporation ( see figures 2 , s2 , and dna sequences for wild - type and mutant ubiquitin ) . ha - ub - alkyne and vme probes were prepared and purified according to literature procedures ( borodovsky et  al . , 2001 , 2002 ; mcgouran et  al . , 2012 )
the ha - ub - alkyne was ligated to the relevant ub aha mutant by cu(i ) catalyzed triazole formation ( rostovtsev et  al . , 2002 ) to afford the corresponding ha - tagged di - ub probes .
hplc purification of all di - ub probes was performed using strong cation exchange chromatography to obtain high purity preparations removing any unconjugated ha - ub - alkyne .
the separation of ub material of hplc fractions was monitored by sds - page and anti - ha western blotting ( figure  3a ) .
the fractions marked with a dotted box were pooled , concentrated , and used for subsequent ub processing enzyme profiling experiments .
representative hplc chromatograms and intact protein maldi - tof ms spectra are shown for the m1 di - ub probe ( figures 3b and 3c ) .
the reactivity of the purified m1 di - ub probe was tested by  incubation with the deubiquitinating enzyme otubain 2 ( otub2 ) alongside the monomeric ha - ub - alkyne probe and visualized by both silver stain and western blotting ( figure  4a ) .
covalent trapping of otub2 with the probes was observed as a characteristic mass shift and shown to be inhibited by the cysteine alkylating agent n - ethylmaleimide ( nem ) . in order to demonstrate the requirement for the presence of a reactive electrophile ,
a proportion of the material was deactivated by reaction with sodium 2-mercaptoethanesulfonate ( mesna ) . inactivated and active m1 di - ub probes were incubated with four different recombinant dubs ( otub2 , uch - l3 , uch - l5 , and usp15 ) and also a cellular extract prepared from hek293 t cells ( figure  4b ) .
all dubs were successfully labeled with the active m1 di - ub probe , but did not react with the inactivated counterpart . incubating the active probe with
lysate resulted in strong labeling of proteins whereas lysate incubation with the inactive probe did not lead to any labeling .
this demonstrates that the electrophilic trap is required for covalent capture of cellular dubs ( figure  4b ) .
furthermore , conjugation through the native c terminus of the proximal ub of the di - ub probe by e3 ligases does not occur to any significant extent under these labeling conditions . to verify site - specific linkage of the ubiquitin moieties within the di - ub probes ,
lc - ms / ms analyses after digestion with trypsin were performed ( figures 5a and s3a  s3f ) .
to assess the effect of introducing a nonnative triazole linker on the conformation of di - ubiquitin , surface plasmon resonance ( spr ) was carried out to compare the k48 di - ub probe to wild - type ( wt ) k48 di - ubiquitin with regards to its binding affinity for otub1 , a dub selective for k48-linked poly - ub chains ( edelmann et  al . , 2009 ) ( figure  5b , left and middle panel ) .
a catalytically inactive mutant ( otub1 c91s ) was used to avoid covalent binding of the probe .
spr analysis indicated kd values of 50  nm ( di - ub wt ) versus 80  nm ( di - ub probe ) , respectively ( figure  5b , right panel ) .
the otub1 inactivation mutation may further stabilize the substrate interaction as compared to its wild - type enzyme ( wiener et  al . , 2013 ) , but appears to be similar between natural di - ub and the di - ub probe . in order to compare differences in the cellular labeling pattern of the eight di - ub probes ,
the ha - ub - vme and ha - ub - alkyne probes , the respective probe and hek293 t cell lysate were incubated and analyzed by sds - page and anti - ha immunoblotting ( figure  6 ) .
interestingly , di - ub probe labeling profiles are distinct from one another and notably so from the mono - ub probes , suggesting a higher dub selectivity . to investigate this in more detail , cell lysate probe labeling followed by immunoprecipitation ( ip ) with anti - ha antibody agarose beads
the elution fractions of all ips were analyzed by tandem mass spectrometry to identify ubiquitin processing enzymes trapped by the probes . in total , 29 dubs were identified by lc - ms / ms in all ten probe ips combined including 18  usps , four uchs , five otus , one mjd , and one sumo deconjugase ( figure  7a ) .
the mass spectrometric intensities of peptide signals for detected dubs in all experiments were subjected to relative quantitation in order to compare dub selectivity for the various ub linkages . in separate experiments ,
poly - ub chains of defined linkage were incubated in  vitro with various recombinant members of the uch , usp and otu families to compare and further explore dub preferences for different ub linkages .
samples were analyzed by sds - page and anti - ub immunoblotting to visualize disassembly of the respective ub chains ( figures 7b7i ) .
as a positive control all recombinant dubs were labeled with ha - ub - vme , demonstrating that all enzymes used in this panel were active ( figure  7j ) .
the monomeric ha - ub - vme and alkyne probes showed generally the lowest labeling specificity across the panel of cellular dubs detected ( figure  6 , lanes 1 and 2 ) , whereas interestingly , the m1 di - ub probe was the one with the most promiscuous specificity among the di - ub probes ( figure  7 , lane 3 ) . it should be noted that although the di - ub probes display the electrophilic trap in the appropriate position to mimic the native isopeptide bond ( figures 1 and s1 ) , the m1 probe is linked via the amino acid side chain ( m1 position ) rather than the protein n terminus .
the different relative position of the distal ubiquitin and the greater degree of flexibility afforded by this side chain linkage in comparison to the native peptide bond apparently cause decreased specificity of this probe ( komander et  al . , 2009 ) .
although many dubs showed little reactivity toward the different di - ub linkage types in comparison to the mono - ub probes , several were selective for particular di - ub probes tested .
we detected all four cysteine protease members of the uch family in our probe ip experiments .
consistent with the observation that uch enzymes are inefficient at ubiquitin chain cleavage ( kulathu and komander , 2012 ; zhou et  al . , 2012 ) , we observed low general reactivity toward the di - ub probes in comparison to the monomeric probes in our ip samples ( figure  7a ) .
we also detected little evidence of cleavage in our recombinant enzyme assays ( figure  7b ) .
with otu6b , we observe little reactivity toward the di - ub probes in comparison to the monomeric probes .
2010 ) , does indeed reflect this specificity in our assay showing the highest reactivity among the di - ub probes , although the specificity we observe is somewhat more modest than that seen within the previously reported in  vitro assays with recombinant enzymes ( mevissen et  al . , 2013 ) .
otub1 displayed k48 linkage - specificity among the di - ub probes ( figure  7a ) , in line with both literature observations ( edelmann et  al .
2010 ) and results of our poly - ub cleavage assay ( figure  7 g ) .
vcip interestingly displayed a preference for the k11 linkage among the di - ub probes in line with in  vitro assays using truncated vcip ( mevissen et  al .
, 2013 ) , although little activity was seen with the k48 di - ub linked probe as would be expected from the in  vitro assays using the truncated form ( mevissen et  al . , 2013 ) .
for usps , it has previously been noted that these enzymes are generally specific to ubiquitination substrates rather than the linkage of the ubiquitin chain , with a low degree of selectivity observed between the various ubiquitin linkages ( faesen et  al . , 2011 ) .
our poly - ub cleavage assays and probe labeling experiments confirm this observation , although we do see some examples of apparent specificity in the reactivity toward different di - ub probes .
for instance , usp13 , usp25 , and usp40 showed high relative affinity for the m1 probe .
interestingly , previous in  vitro assays suggested little reactivity with linear ub chains for these dubs ( faesen et  al . , 2011 ; kulathu and komander , 2012 ) .
this discrepancy could be due to either the artificial nature of the in  vitro assay or the greater flexibility of the linkage present in our m1 di - ub probe as compared to the native peptide bond ( komander et  al . , 2009 ) .
interestingly , otulin , a dub exclusively specific for cleaving linear poly - ub chains ( keusekotten et  al . ,
2013 ) , was not detected with the m1 di - ub probe , possibly due to similar reasons .
usp15 in our ip results showed a degree of selectivity for k27-linked probe ( figure  7a ) .
the corresponding poly - ub cleavage assay showed that usp15 in its recombinant form was indeed active in cleaving k27 chains , but also other linkages ( figures 7b7i ) .
usp16 showed a high relative reactivity toward the k27 and to  a lesser extent the k29 probe in our assay .
literature precedence suggests that usp16 is active against these chain types although a less striking selectivity was observed previously ( faesen et  al . , 2011 ) .
di - ub probe labeling data also suggested k11 selectivity by usp5 ( figure  7a ) , whereas our poly - ub cleavage data suggested a very low degree of reactivity generally ( figures 7b7i ) .
previous data has , however , shown usp5 activity against k11 chains ( bremm et  al . , 2010 ) .
usp47 was found to have a high degree of specificity toward k27 linkages based on reactivity toward the di - ub probes and the recombinant dub assays , and usp24 was highlighted to have a high degree of specificity toward the k27 and k11 probes .
we also detected ataxin 3 , principally in the m1 and k29 probe ips despite previous suggestions that it has low activity toward chains of four ubs or less ( mao et  al .
ub fusion protein 1 was also detected , and the highest relative abundance was observed with the m1 di - ub probe .
brca1-a showed little selectivity among the different probes . taken together , our engineered probes representing all naturally occurring di - ub linkages provide clues on topology preferences of cellular dubs for different poly - ub chains , some of which were validated in  vitro .
interestingly , many dubs appear also to recognize noncanonical ub - linkages other than k48/63 scaffold , suggesting the processing of a wider range of ubiquitinated substrates with different ub linkage topologies . quantitative mass spectrometry analysis of di - ub probe ip material also revealed components of the ub conjugation machinery were enriched by the different di - ub probes , as has been previously observed with mono - ub probes ( love et  al . , 2009 ) .
a panel of conjugating enzymes was detected including those from hect and rbr e3 families along with ring e3 ligases and e2 family members ( table s2 ) .
the application of engineered activity probes based on di - ub scaffolds allows us to address dub ub - linkage specificity in a cellular context , which generally appears less biased toward canonical ub - linkages than previously anticipated .
certain dubs , in particular otus , retain selectivity for  distinct poly - ub chains as predicted using recombinant enzyme assays . the approach described provides insights into the selectivity of dubs within the cognate environment of substrates as part of multiprotein complexes , which is often problematic to reconstitute with in  vitro experiments .
the detection of many enzymes of the ubiquitin conjugation machinery is noteworthy and may also provide us with a useful tool to study such enzymes in a cellular environment .
these advances provide a framework for further understanding the role of dubs and ub conjugating enzymes in cellular functions under normal physiological conditions as well as in disease
. a greater understanding of dub ub - linkage specificity may also offer inroads to assess cellular effects and specificity of small molecule inhibitors for future pharmacological intervention strategies .
reagents were purchased from aldrich and used as supplied . deionized water ( minimum resistivity 18.2 m ) was used for preparation of buffers and chemical and biochemical reactions .
precision plus protein standard all blue gel marker ( biorad ) was used for sds - page .
ha - ub - alkyne , ha - ub - vme probes , and azidohomoalanine were synthesized in house according to literature procedures ( borodovsky et  al . , 2002 ; link et  al . , 2007 ; mcgouran et  al . , 2012 ) using a ha - ub(1 - 75)-intein - chitin
t cells were cultured in dmem ( dulbecco ) medium supplemented with 10% fcs ( sigma ) and 1% penicillin / streptomycin at 37c with 5% co2 . following overnight culture bacteria were harvested and the wild - type plasmid dna containing the ubiquitin sequence cloned into the pet15b ( addgene ) vector was isolated , using wizard plus sv minipreps dna purification system ( promega ) according to the manufacturer s protocol .
dna concentration was analyzed by measuring absorption at 260  nm wavelength using a nanodrop spectrophotometer .
point mutants of ubiquitin were created using the quickchange ii site directed mutagenesis kit by stratagene .
mutant i2a was used as template for all following substitutions : k6 m , k11 m , k27 m , k29 m , k33 m , k48 m , and k63 m .
the primers used for the design of the ubiquitin mutants are listed in table s1 .
mutant constructs were verified by dna sequencing ( source bioscience , department of biochemistry , university of oxford ) .
sequencing results can be found in the dna sequences for wild - type and mutant ubiquitin .
for the expression of wt and mutant ub , methionine auxotroph b834(de3 ) competent cells were used ( novagen ) according to manufacturers protocol .
glycerol stocks were prepared by picking a single colony from an agar plate and incubating it in lb medium with shaking ( 160  rpm ) at 37c for 18  hr . a 100  l cell suspension was mixed with 900  l of 60% glycerol in water and stored at 80c .
bacteria were incubated in selenomet medium ( molecular dimensions ) containing 40  g / ml methionine at 37c for 18  hr . after transferring the bacteria to up to 500  ml selenomet medium containing and methionine ,
bacteria were grown with shaking ( 180  rpm ) at 37c until they reached an  od600 of 0.9 .
after harvesting , the cells were washed two times with methionine - free selenomet medium before resuspension in selenomet medium containing 40  g / ml azidohomoalanine ( aha ) and 0.4  mm iptg .
protein expression was accomplished at 37c for 4  hr with continuous shaking ( 180  rpm ) .
subsequently the harvested bacteria were resuspended in lysis buffer ( 100  mm tris ph 7.5 , protease inhibitors [ roche ] , 0.02%  np40 , 0.4  mg / ml lysozyme ) before 1.2  mg / ml mgso4 and 12  g / ml dnasei were added .
vigorously vortexed cell lysate was cleared by centrifugation ( 16,000   g , 30  min , 4c ) and treated with 0.5% perchloric acid on ice .
remaining ub in the supernatant was dialyzed in 50  mm naoac  ph 4.5 for 18  hr at 4c .
the ub mutants were resolved on an 18% bis - tris gel and visualized by silver staining .
linear mode maldi - tof ms analysis ( bruker ) was also performed ( figure 2 ) .
a further aliquot of each mutant was subjected to digestion by trypsin and the resulting peptides analyzed by lc - ms / ms ( mackeen et  al . , 2010 ) to confirm aha incorporation . in brief
, proteins were resuspended in 6  m urea , reduced with dithiothreitol and alkylated with iodoacetamide before proteolysis with trypsin .
desalted samples were separated on a 75  m  250  mm reversed phase nano - acquity - uplc ( waters ) and analyzed by a coupled q - tof premier mass spectrometer ( waters ) .
ms / ms spectra were searched against a human database containing the ub mutant sequences ( 86,755 sequences ) in mascot ( matrix science v2.4 ) allowing one missed cleavage and 50 ppm/0.1  da mass deviation in ms and ms / ms , respectively .
oxidation of methionine , methionine to azidohomoalanine exchange , and deamidation of asparagine and glutamine were used as variable modifications .
ms / ms spectra showing incorporation of aha in the positions m1 , k6 , k11 , k27 , k29 , k33 , k48 , and k63 are shown in figures s2a  s2h .
hplc - purified ha - ub - alkyne ( 77  l , 2  mg / ml ) was mixed with the appropriate azidohomoalanine incorporated ub mutant ( 805  l , 0.56  mg / ml ) in 50  mm na2hpo4/nah2po4 ph 8 buffer .
tris[(1-ethoxycarbonylmethyl-1h-1,2,3-triazol-4-yl)-methyl]amine ( 90  g in 15  l ch3cn ) and cu(i)br ( 120  g in 15  l ch3cn ) were added before shaking at room temperature .
after 30  min , a second portion of cu(i)br ( 120  g in 15  l ch3cn ) was added , prior to a further 30  min incubation with shaking .
the di - ub probe was buffer exchanged into 50  mm naoac ph 4.5 using gel filtration ( pd-10 , ge healthcare ) .
the resulting probes were hplc ( agilent 1100 ) purified using a scx biomonolith column ( 5.2   4.95  mm , agilent ) with a linear gradient from 0% to 100% buffer b , 50  mm naoac ( ph 4.5 , buffer a ) , 50  mm naoac ( ph 4.5 ) , and 1  m nacl ( buffer  b ) at a flow rate of 0.5  ml / min .
product - containing fractions were identified based on sds - page separation and anti - ha immunoblotting ( figure  3 ) and concentrated .
the correct linkage was confirmed by lc - ms / ms analysis for seven out of the eight di - ub probes ( figures 5a and s3a  s3f ) .
a minority of purifications , most notably for k33 di - ub , contained tri - ub species possibly resulting from insufficient cleavage of the n - terminal azidohomoalanine leading to double ligation . these by - products were removed .
one hundred nanograms of recombinant dubs otub1 , otub2 , uch - l1 , uch - l3 , uch - l5 , bap1 , usp2 , usp5 , usp7 , usp8 , usp15 , usp20 , usp21 , usp28 , and usp47 ( all obtained from progenra ) or 20  g hek293 t cell lysate , prepared as described ( mcgouran et  al . ,
labeling was conducted in presence or absence of 10  mm n - ethylmaleimide ( nem ) to confirm specificity .
volumes were adjusted to 15  l with 50  mm nah2po4/na2hpo4 ph 8.0 buffer .
all reactions were incubated at 37c for 3  hr followed by addition of 5  l of 3 reducing sample buffer and heating to 95c for 5  min .
samples were resolved on a nupage 4%12% bis - tris gradient gel and visualized by silver staining or western blotting methods using a directly coupled anti ha - hrp antibody ( 1:10,000 dilution , sigma aldrich h6533 ) .
an inactivated linear di - ub probe was also generated to discount the possibility of conjugation through the di - ub c terminus .
two micrograms of linear di - ub probe was incubated in 10  l inactivation buffer ( 50  mm mesna , 50  mm nah2po4/na2hpo4 ph 8.0 ) at 37c for 24  hr . a total of 1.5  l of 50  mm mesna was added along with 10  l 50  mm nah2po4/na2hpo4 ph 8.0 and di - ub probe was incubated for a further 40  hr at room temperature ( rt ) .
the buffer was exchanged to 50  mm nah2po4/na2hpo4 ph 8.0 by centrifugal filter devices .
alternatively , 1  m recombinant dubs were incubated with 100  ng of poly - linked ub chains ( k6/k11/k27/k29/k33/k48 , or k63 , biomol ) for 3  hr at 37c in 50  mm tris ( ph 8.0 ) and 1  mm dtt , separated by nupage 4%12% bis - tris gradient gel electrophoresis , and analyzed by anti - ub immunoblotting ( 1:3,000 dilution , bd pharmingen ) .
surface plasmon resonance experiments were performed on a biacore 2000 instrument equipped with research grade cm5 sensor chips ( biacore ab ) .
all surfaces were prepared by an amine - coupling method ( johnsson et  al . , 1991 ) using reagents available from biacore ab ( n - ethyl - n-dimethylaminopropyl - carbodiimide [ edc ] , n - hydroxysuccinimide [ nhs ] , and 1  m ethanolamine - hcl ) .
flow cells were activated for 15  min at 5  l / min with a solution containing 50  mm nhs and 0.2  mm edc .
k48 di - ub wt and k48 di - ub probe were diluted to 20  g / ml in 10  mm sodium acetate ( ph 4.5 ) and injected until immobilization levels of 12,00014,000 ru were achieved .
the remaining reactive groups were blocked with a 12  min injection of 1  m ethanolamine - hcl at ph 8.5 .
otub1 ( c91s mutant ) binding data were collected at 20c at a flow rate of 5  l / min in a solution of running buffer ( 10  mm hepes [ ph 7.4 ] , 150  mm nacl , 3  mm edta , and 0.005% p20 ) for both reference and reaction surfaces . otub1 c91s was dissolved directly in the running buffer at a stock concentration of 100  m .
serial dilutions were made in running buffer across the concentration series from 10  nm to 3  m . samples were injected for 300  s followed by re - equilibration with running buffer .
the raw response data were zeroed on both the response and time axes prior to the start of the injection . to correct for bulk refractive index changes , responses from a reference surface without protein were subtracted from the reaction surface data . to extract equilibrium dissociation constants
, we plotted binding responses at equilibrium against ligand concentration and measured concentration values at 50% site occupation ( figure  5b ) .
preparation of cell lysate and labeling of endogenous active dubs using the ha - ub - alkyne , ha - ub - vme , and ha - di - ub probes was performed according to literature procedures ( borodovsky et  al . , 2002 ) .
for the quantitative mass spectrometry experiments , 1  mg of hek293 t cell lysate was incubated with 20  g of di - ub probe ( or 10  g of the mono - ub probe ) in net buffer ( 50  mm tris ph 7.5 , 5  mm edta , 150  mm nacl , 0.5% np-40 ) in a total volume of 180  l for 3  hr at 37c .
sds was then added to 0.5% final concentration and the protein mixture was incubated at 4c for 1  hr .
the sds concentration was reduced to 0.05% by adding net buffer before the mixture was added to monoclonal anti - ha agarose beads ( sigma aldrich ) .
immunoprecipitation was carried out at 4c for 18  hr followed by three washing steps with high salt net - buffer ( 50  mm tris ph 7.5 , 5  mm edta , 300  mm nacl , 0.5% np-40 ) .
elution was accomplished by addition of ha peptide to a final concentration of 3  mg / ml .
the immunoprecipitated eluate was desalted using chloroform - methanol extraction , followed by overnight in - solution tryptic digestion and subsequent desalting as described previously ( altun et  al . , 2011 ) . in brief , proteins were chloroform - methanol precipitated prior to resuspension in 6  m urea , reduced with dithiothreitol , and alkylated with iodoacetamide before proteolysis with trypsin .
tryptic digests were desalted and subjected to lc - ms / ms ( 75  m  250  mm reversed phase nano - acquity - uplc , waters ) analysis using a thermo ltq orbitrap velos ( 30,000 resolution , top 20 , collision - induced dissociation ) workflow and a gradient of 1%40% acetonitrile in 48  min at a  flow rate of 250 nl / min .
peptides were  detected and quantified with progenesis lc - ms software ( version 3.1.4003.30577 ) using default settings ( no deconvolution / deisotoping , 200 most intense ms / ms peaks ) .
a merged peak list generated by progenesis lc - ms was searched against the uniprot human database ( version.3.80 , 20,339 entries ) using mascot v2.4 , allowing one missed cleavage and 20 ppm/ 0.5  da mass deviations in ms and ms / ms .
oxidation of methionine and deamidation of asparagine and glutamine were used as variable modifications . for label - free protein quantitation
raw protein abundance values from progenesis lc - ms were first normalized using quantile normalization and technical replicates aggregated by taking the median normalized value for each protein in each sample .
quantitation of enzyme abundance based on the different probe immunoprecipitations was based on at least two unique peptides , confidence score > 50 and anova ( p value ) < 0.05 in order to generate the heat maps for figure  7a .
 summaryposttranslational modification with ubiquitin ( ub ) controls many cellular processes , and aberrant ubiquitination can contribute to cancer , immunopathology , and neurodegeneration . 
 the versatility arises from the ability of ub to form polymer chains with eight distinct linkages via lysine side chains and the n terminus . in this study , we engineered di - ub probes mimicking all eight different poly - ub linkages and profiled the deubiquitinating enzyme ( dub ) selectivity for recognizing di - ub moieties in  cellular extracts . 
 mass spectrometric profiling revealed that  most dubs examined have broad selectivity , whereas a subset displays a clear preference for recognizing noncanonical over k48/k63  ub linkages . 
 our results expand knowledge of ub processing enzyme functions in cellular contexts that currently depends largely on using recombinant enzymes and substrates . 
Introduction
Results and Discussion
Significance
Experimental Procedures