adp - ribose ( adpr ) is a post - translational 
 modification that is 
 synthesized by a family of adp - ribosyltransferases , commonly known as poly(adp - ribose ) polymerases , or parps . 

these modifications are derived from the hydrolysis of nad and exist in both the monomeric and polymeric forms , the latter 
 of which is made up of 2200 adpr subunits .
the canonical role 
 for this polymer has been in the identification and repair of dna 
 nicks and double - stranded breaks via activation of the founding member 
 of the parp family , parp-1 . indeed ,
this 
 role has ushered in parp-1 as a chemotherapeutic target , as the loss 
 of parp-1 sensitizes cells to genomic assault by established chemotherapeutic 
 and radiation - based treatment .
it is worth 
 noting , however , that par s cellular role has expanded beyond 
 dna repair into regulation of ( among others ) apoptosis , chromatin structure , synthesis of dna / rna , telomere maintenance , protein degradation , and microrna activities .
not surprisingly , 
 the increase in understanding of par s biological roles has 
 led to recognition of its therapeutic potential beyond modulation 
 of dna damage , including the treatment of necrosis and inflammation .
par s relative , mono(adp - ribose ) , is 
 far less studied but has received increasing attention due to a number 
 of recent studies that have identified the enzymes which reverse mono(adp - ribosylation ) 
 as well as novel roles for mono(adp - ribose ) in the cell . in an effort to aid in the understanding of 
 the mono- and poly(adp - ribosyl)ated proteome , we have looked to mass 
 spectrometry to define the molecular basis of adp - ribosylation and 
 will begin by characterizing the poly(adp - ribosylation ) ( or parylation ) 
 activity of human parp-1 ( hparp-1 ) .
the hurdles that have kept 
 mass spectrometry and proteomics from 
 becoming universal tools for studying parylation have to do with the 
 physical properties of the modification itself : first , the modification 
 can expand linearly or by branching and vary dramatically in length , 
 resulting in a large , heterogeneous polymer without a defined mass . 

second , many of the amino acid attachment sites are base - labile , preventing researchers from exposing the modified 
 proteins or peptides to basic solutions , which are commonly used in 
 proteomic sample preparations . finally , the modification is dynamic , 
 with basal levels existing below the level of detection of most molecular 
 tools used in proteomics .
one recently published approach to identify 
 adp - ribosylation sites by mass spectrometry paired boronate enrichment 
 of adp - ribosylated proteins with subsequent release of mono- and poly(adp - ribose ) 
 from substrates by hydroxylamine .
this 
 elution strategy breaks ester bonds between the adpr subunits and 
 the carboxyl groups of aspartate and glutamate residues , leaving a 
 characteristic 15.01 da mass signature on the modified residue .
notably , 
 this approach can not identify nonacidic adp - ribosylated residues and 
 up to 33% of total adp - ribosylated amino acid residues have been shown 
 to be hydroxylamine - insensitive .
in particular , 
 lysine residues are important for the in vitro and in vivo activation 
 of parp-1 as well as substrate regulation by parps , 
 for example , chromatin remodeling via parylation of the lysine residues 
 on histone tails . because a global 
 approach to identify all possible adp - ribosylation 
 sites is still needed , we have developed an enrichment protocol based 
 on the digestion of adpr by snake venom phosphodiesterase ( svp ) , a 
 pyrophosphatase that cleaves adpr subunits down to phospho(ribose ) 
 and 5-amp .
this digestion produces 
 a single phospho(ribose ) group at the site of modification that can 
 be identified by mass spectrometry as an adduct of 212.01 da .
given the similarity of phospho(ribosyl ) and 
 phosphate groups , we reasoned that existing phosphoproteomic techniques 
 may be used to enrich phospho(ribosyl)ated peptides .
indeed , a 2010 
 phosphoenrichment study that utilized immobilized metal affinity chromatography 
 ( imac ) to enrich phosphopeptides was searched in 2012 for a coenrichment 
 of adpr or phosphoribose , both of which were found to have been enriched .
more recently , chapman et al . demonstrated the 
 feasibility of this approach to identify parylation sites on a purified , 
 automodified human parp-1 .
here we have 
 independently tested and validated this approach to identify adp - ribosylation 
 sites ; we further compared three commercially available phosphoenrichment 
 matrices and their use in enriching and characterizing phospho(ribosyl)ated 
 peptides of hparp-1 from a complex background of hela whole cell lysate . 

finally , we have demonstrated the application of this method to identify 
 endogenous mono- and poly(adp - ribosyl)ation sites by mass spectrometry , 
 yielding both known and novel acceptors of adpr , including a number 
 that identify adpr on arginine residues .
, 6 l of his - parp-1 expressing de3 cells was lysed in a cell 
 homogenizer in the presence of 0.1% np-40 , 20 u / ml dnase i , 5 mm mgcl2 , 1 m bestatin , 1 m pepstatin a , and 1 
 roche complete edta - free protease inhibitor .
lysate was cleared by 
 centrifugation and loaded onto an kta fplc ( ge , 18 - 1900 - 26 ) 
 with a pre - equilibrated 5 ml histrap ff crude column ( ge , 17 - 5286 - 01 ) , 
 where it was washed with 10 column volumes of loading buffer ( 20 mm 
 sodium phosphate ph 7.4 , 1 m nacl , 0.5 mm tcep , 40 mm imidazole ph 
 7.4 , 1% glycerol , 1 roche complete edta - free protease inhibitor ) 
 before being eluted in 2 column volumes of elution buffer ( 20 mm sodium 
 phosphate ph 7.4 , 0.5 m nacl , 0.5 mm tcep , 0.5 m imidazole ph 7.4 , 
 1% glycerol ) .
eluted samples were diluted 1:1 in heparin no - salt buffer 
 ( 50 mm tris ph 7.0 , 0.1 mm tris(2-carboxyethyl)phosphine , 1% glycerol ) 
 and loaded onto a pre - equilibrated 5 ml heparin column ( ge , 17 - 0407 - 01 ) , 
 washed with 5 volumes of low - salt buffer ( 50 mm tris ph 7 , 0.1 mm 
 tcep , 250 mm nacl ) and eluted over a gradient from 0 to 70% high - salt 
 buffer ( 50 mm tris ph 7 , 0.1 mm tcep , 1 m nacl , 1% glycerol ) .
desired 
 fractions were pooled and concentrated using a spin concentrator ( 30  000 
 mwco , amicon z648035 ) before being loaded onto a pre - equilibrated 
 size exclusion column ( ge , superdex 200/10/300 gl ) in size purification 
 buffer ( 25 mm hepes ph 8 , 0.1 mm tcep , 150 mm nacl ) ; desired fractions 
 were pooled and stored at 80 c .
brief , two 100 unit vials of crotalus adamanteus phosphodiesterase 
 i ( worthington , ls003926 ) were dissolved into 1 ml of loading buffer 
 ( 10 mm tris - cl ph 7.5 , 50 mm nacl , 10% glycerol ) and then loaded onto 
 a pre - equilibrated 1 ml hitrap blue sepharose column ( ge , 17 - 0412 - 01 ) , 
 washed with 5 column volumes of loading buffer and then 5 column volumes 
 of elution buffer ( 10 mm tris - cl ph 7.5 , 50 mm nacl , 10% glycerol , 
 150 mm potassium phosphate ) .
desired fractions were pooled , dialyzed 
 against loading buffer , and stored at 80 c . if enzyme 
 preps were to be used to treat denatured proteins , an additional purification 
 was needed to remove any contaminating proteases : samples were dialyzed 
 into size exclusion chromatography buffer ( 10 mm tris - cl ph 7.3 , 50 
 mm nacl , 15 mm mgcl2 , 1% glycerol ) and resolved over a 
 superdex 200/10/300 gl ( ge healthcare ) using an kta fplc ( ge , 
 18 - 1900 - 26 ) ; desired fractions were pooled and stored at 80 
 c .
forward 
 ( gggtggcggccgcttggg ) and reverse ( cccaagcggccgcaaccc ) 
 oligos were mixed 1:1 in h2o , heated to 95 c for 
 2 min , and then ramp - cooled to 25 c over 45 min .
hisparp-1 was 
 attached to promega magnehis beads ( 1 g parp-1/l beads/5 
 l attachment buffer ) for 2 h at 4 c in attachment buffer 
 ( 50 mm tris ph 7.4 , 1% tween , 0.2 mm dtt , 10% glycerol , 10 mm mgcl2 ) .
beads were washed twice with 100 l of wash buffer 
 ( 50 mm sodium phosphate buffer ph 7.4 , 200 mm nacl , 5 mm imidazole 
 ph 7.4 ) and then exposed to 30 m ( 0.6% hot ) p 
 -nad+ in automodification buffer ( 20 mm tris ph 7.5 , 50 mm 
 nacl , 50 m tcep , 5 mm mgcl2 , 1.2 m annealed 
 dna ) for 10 min , followed by a chase of 2 mm cold -nad+ for 
 60 min , all at 25 c rotating at 500 rpm . for sds - page , beads 
 were washed twice in 100 l of wash buffer and eluted into 15 
 l of 1 sds - page buffer , separated on an in - house 610% 
 sds - page gel , fixed overnight ( 50% methanol , 10% acetic acid ) , washed 
 for 30 min ( h2o ) , stained with pro - q diamond phosphoprotein 
 stain ( invitrogen , mp 33300 ) for 1 h , destained for 3  30 min 
 ( 20% acetonitrile , 50 mm sodium acetate ph 4 ) , washed for 10 min ( h2o ) , and imaged on a fuji fla7000 ( filter : o580 , wavelength : 
 532 nm ) .
pro - q diamond staining was validated based on comparison 
 to pro - q diamond peppermintstick ladder ( life technologies , p27167 ) . 

total protein was determined by coomassie blue staining ( invitrogen 
 lc6060 ) and p - labeling was determined by overnight exposure 
 against a phosphor - screen ( ge , bas - iii 2040 ) , followed by imaging 
 on a fuji fla7000 ( ip ) .
western blotting for poly(adp - ribose ) was 
 performed by transferring proteins ( invitrogen xcell ii blot module ) 
 from an in - house 610% sds page gel to a nitrocellulose membrane 
 ( bio - rad ) , and membranes were blocked in 5% milk in pbs before being 
 incubated in primary antibody ( anti - par , clone lp-9610 from bd biosciences ) 
 for 1 h at room temperature , rinsed in pbs - t , and then incubated in 
 secondary antibody ( anti - rabbit 800 nm from li - cor biosciences ) for 
 1 h before being imaged on an odyssey clx and analyzed in image studio 
 ( from li - cor , version 2.0 ) .
one g 
 of parylated hisparp-1 was treated with 500 munits of purified svp 
 in svp digestion buffer ( 50 mm tris ph 7.5 , 150 mm nacl , 15 mm mgcl2 , 20 mm 3-aminobenzamide ) for 2 h at 25 c , 500 rpm hparp-1 was induced to automodify in vitro ( as previously described ) 
 and mixed in a 1:2 ratio with bsa , and 1 g hparp1/2 
 g bsa was aliquoted and exposed to 5% ammonium hydroxide , 500 
 mm kh2po4 ph 7 , or automodification buffer ( control ) 
 in a total volume of 10 l for 5 min .
reactions were quenched 
 by adding 1 ml of ice - cold precipitation buffer ( 0.02% deoxycholate , 
 4% triton x-100 , 10% tca ) and stored at 20 c for 2 h 
 before being pelleted by centrifugation at 4 c and decanted . 

pellets were washed with ice - cold acetone containing 20 g / ml 
 glycogen as a carrier , stored at 20 c for 30 min , pelleted , 
 decanted , dried by speedvac , and resuspended in sds running buffer . 
 for sds - page analysis , an equal volume of 2 sds - page buffer 

was added to samples for analysis on an in - house 610% tris - glycine 
 gel .
hela cells ( kyoto ) were grown in arginine 
 and lysine free dmem ( pierce ) containing 10% dialyzed fbs ( sigma ) , 
 0.4 mm arginine ( c6n4 from cambridge , c6n4 from sigma ) , and 0.8 mm lysine ( c6n2 from isotec , c6n2 from sigma ) .
trophoblast stem cells from parg knockout 
 mice ( e3.5 from 129.svj mice , acquired from dr .
david koh of johns 
 hopkins university ) were grown in arginine- 
 and lysine - free rpmi 1640 ( pierce ) containing 16% dialyzed fbs ( sigma ) , 
 0.4 mm arginine ( c6n4 from cambridge , c6n4 from sigma ) , 0.8 mm lysine ( c6n2 from isotec , c6n2 from sigma ) , 1 mm sodium pyruvate ( life technologies ) , 
 2 mm l - glutamine ( life technologies ) , 25 units / ml penicillin 
 ( cellgro ) , 25 units / ml streptomycin ( cellgro ) , 100 m monothioglycerol 
 ( sigma ) , 1 g / ml heparin sulfate , 25 ng / ml fgf-4 , and 0.5 mm 
 benzamide ( sigma ) .
all cells were treated with 5 mm n - methyl - n-nitro - n - nitrosoguanidine ( mnng , from 
 accustandard ) for 5 min before being washed three times with ice cold 
 pbs ( gibco ) and lysed in either 6 m guanidine - hydrochloride ( sigma ) , 
 8 m urea ( sigma ) or lysis buffer ( 50 mm tris ph 7.5 , 0.4 m nacl , 1 
 mm edta , 1 edta - free complete protease - inhibitor from roche , 
 1% np-40 , 1 g / ml adp - hpd , and 0.1% sodium deoxycholate ) .
cells 
 lysed in either guanidine - hydrochloride or urea were subjected to 
 sonication in an ice bath for 10 min with 30 s breaks between 30 s 
 cycles ( bioruptor standard ) .
two 
 mg of whole cell 
 lysate in 1 lysis buffer ( 50 mm tris ph 7.5 , 0.4 m nacl , 1 mm 
 edta , 1 edta - free complete protease - inhibitor from roche , 1% 
 np-40 , 1 g / ml adp - hpd , and 0.1% sodium deoxycholate ) was incubated 
 at 5 mg / ml with 40 l of macrodomain - conjugated agarose beads 
 ( tulip # 2302 ) at 4 c overnight before being washed three times 
 with wash buffer ( 50 mm tris ph 7.5 , 0.4 m nacl , and 0.1% sodium deoxycholate ) 
 and eluted by 8 m urea ph 7 .
all proteins were denatured in 8 m urea 
 ph 7 for 10 min at 37 c before being reduced in 1 mm tris(2-carboxyethyl)phosphine 
 ( life technologies ) for 10 min and then alkylated in 2 mm 2-chloroacetamide 
 ( sigma ) for 10 min in the dark .
if automodified hparp-1 is to be added 
 as a standard , it is prepared the same way and added to the lysate 
 background at this point .
samples were then diluted to a final concentration 
 of 1 m urea , 50 mm nacl , 15 mm mgcl2 , 1 mm cacl2 , and 0.2 m tris ph 7.3 .
five g of purified svp were added 
 for each mg of whole cell lysate and incubated for 2 h at 37 c .
samples in 1 m urea , 
 0.2 m tris - cl ph 7.3 , 1 mm cacl2 , 15 mm mgcl2 , and 50 mm nacl are treated with endoproteinase lysc ( wako ) 1:50 
 enzyme / substrate .
after 1 h , trypsin ( sigma ) was added at a 1:50 enzyme / substrate 
 ratio , and the entire reaction was incubated overnight .
reaction was 
 stopped by adding an equal volume of desalting solvent a ( 5% acetonitrile , 
 0.1% tfa ) and desalted on a c18 stagetip and eluted in desalting solvent 
 b ( 80% acetonitrile , 0.1% tf ) as in rappsilber et al .
hela cells were scraped into 6 m gnd - hcl , 
 lysed by sonication , and cleared by centrifugation .
300 g of 
 protein was then reduced , alkylated , and in - solution digested by lysc 
 and trypsin , as described in  in - solution protein digestion  . 

to this mixture of peptides , 30 g of peptides from automodified , 
 svp - treated hparp-1 and 10 g of peptides from bovine casein 
 were added .
this mixture was then sampled as input and split into 
 3 equal volumes that were enriched by either imac ( sigma phos - select 
 beads ) or moac ( gl sciences or glysci tips containing zirchrom tio2 beads ) .
2008 ; in brief , they were incubated for 1 h , shaking 
 at 25 c , on 50 l of phos - select beads in binding buffer 
 ( 0.1% formic acid , 40% acetonitrile ) .
these beads were then transferred 
 to a pre - equilibrated stagetip , where 
 they were washed with binding solvent three times , acidified with 
 1% fa , and eluted onto the stagetip with 0.5 m potassium phosphate 
 ph 7 , where they were acidified with 1% fa again and washed with desalting 
 solvent a ( 5% acetonitrile , 0.1% tfa ) .
they were then eluted with 
 desalting solvent b ( 80% acetonitrile , 0.1% tfa ) .
moac samples were 
 enriched by either gl sciences or glysci tio2 tips , both 
 by their manufacturer s protocols with the adaptation that 
 they were eluted with 0.5 m potassium phosphate ph 7 .
peptides were separated 
 on a thermo - dionex rslcnano uplc instrument with 10 cm  
 75 m i d fused silica capillary columns with 10 m 
 tip opening made in - house with a laser puller ( sutter ) and packed 
 with 3 m reversed phase c18 beads ( reprosil-c18.aq , 120  , 
 dr . maisch ) with a 90 min gradient of 335% b at 200 nl / min . 

liquid chromatography ( lc ) solvent a was 0.1% acetic acid and lc solvent 
 b was 0.1% acetic acid , 99.9% acetonitrile .
data - dependent analysis was applied 
 using top5 selection , and fragmentation was induced by cid and hcd . 

raw 
 files were analyzed by maxquant version 1.4.0.8 using protein , 
 peptide , and site fdrs of 0.01 and a score minimum of 40 for modified 
 peptides and 0 for unmodified peptides and delta score minimum of 
 17 for modified peptides and 0 for unmodified peptides .
sequences 
 were searched against the uniprot human database ( definitions updated 
 may 29 , 2013 ) .
endogenous phospho- and phosphoribose peptide lists 
 were further restricted by a delta ppm of 2 from each 
 respective data set ( average and standard deviation were calculated 
 from the complete tandem mass spectra ( ms / ms ) list of identified peptide 
 precursors ) and the expected heavy / light ratios ( 1 for heavy 
 or light data sets , respectively ) .
maxquant search parameters : variable 
 modifications included oxidation ( m ) , acetylation ( protein n - term ) , 
 phosphorylation ( sty ) , and phosphoribosylation ( dekr ) .
phosphoribosylation 
 ( dekr ) allowed for neutral losses of h3po4 ( phosphoric 
 acid , 97.98 da ) and c5h9po7 ( phosphoribose , 
 212.01 da ) .
max - labeled 
 amino acids were 3 , max missed cleavages were 2 , enzyme was trypsin / p , 
 max charge was 7 , multiplicity was 2 , and silac labels were arg10 
 and lys8 .
as a model for protein parylation we utilized hparp-1 , a poly(adp - ribose ) 
 polymerase capable of autopoly(adp - ribosyl)ation .
exposure of hparp-1 
 to p - labeled -nad+ resulted in an increase in 
 molecular weight of hparp-1 above its unmodified mass of 113 kda , 
 which was correlated with the p signal observed in the 
 autoradiograph , indicating incorporation of p - adpr via 
 hparp-1 automodification ( figure 1a , b , lane 
 1 vs lane 2 ) . upon treatment with svp ( lane 3 ) , the majority of the 
  smear  was lost by both coomassie blue and p detection with an accompanied increase in the intensity of the 
 coomassie - stained band at the expected size of unmodified hparp-1 . 

this result demonstrates svp s ability to break down the polymer 
 at pyrophosphate bonds , potentially reducing the polymer entirely 
 to the phospho(ribosyl ) group on the modified amino acid residue of 
 parylated proteins . visualizing phospho(ribose )
( a  c ) 
 hparp-1 
 automodified in vitro upon exposure to p - labeled nad+ , 
 par formation is evidenced by the p - labeled smear above 
 unmodified hparp-1 ( arrowheads ) . upon treatment with svp , the smear 
 diminishes while the native - sized hparp-1 band reappears .
staining 
 with the phosphostain pro - q diamond indicates that this band is carrying 
 phospho - groups , likely phosphoribose .
( d , e ) pro - q positive product 
 of svp - treated automodified hparp-1 is susceptible to calf intestinal 
 phosphatase ( cip ) treatment . because of the similarity of phospho(ribose ) and phosphate 
 groups , 
 we posited that the phospho(ribosyl)ated hparp-1 might share properties 
 with phosphoproteins . to test this hypothesis , we used the phosphoprotein 
 gel stain , pro - q diamond , to stain the polyacrylamide gel in figure 1a ( figure 1c ) .
while unmodified 
 hparp-1 and modified hparp-1 were weakly stained with pro - q diamond , 
 the signal was significantly increased for svp - treated hparp-1 ( figure 1c , lanes 13 ) .
the phospho - specificity of 
 the dye was confirmed with the two phosphoprotein controls , ovalbumin 
 and -casein , in the protein molecular weight ladder ( figure 1c , marker ) . to confirm that the staining associated 
 with svp - treated hparp-1 is due to the presence of phosphate groups
as 
 expected , upon removal of the phosphate groups by cip , the resultant 
 ribosylated hparp-1 was no longer stained by pro - q diamond ( figures 1d , e ) .
these data suggest that svp treatment of parylated 
 substrates produces phospho(ribose ) groups and that the resultant 
 phosphate groups may have similar physicochemical properties to phosphate 
 groups in phosphoproteins .
we then sought to examine our ability to 
 enrich these phospho(ribose ) groups using phosphopeptide enrichment 
 strategies .
popular phosphoproteomic approaches use immobilized metal affinity 
 chromatography ( imac ) or metal oxide affinity chromatography ( moac ) 
 to enrich phosphopeptides , followed by elution with ammonium hydroxide . 

unfortunately , ammonium hydroxide is highly basic and therefore releases 
 adpr from glutamic and aspartic acid residues . for this reason
, we considered an alternative elution condition , 
 neutral phosphate buffer , which has been used previously to competitively 
 elute phosphopeptides . to assess adpr 
 stability in the presence of phosphate buffer p - labeled , 
 automodified hparp-1 was exposed to either 5% nh4oh , 0.5 
 m kh2po4 ph 7 or control ( automodification buffer 
 containing 20 mm tris ph 7.5 ) .
as can be seen in figure 2 , both the control and the neutral phosphate buffers maintained 
 hparp-1 in its parylated form ( smeared ) while ammonia hydrolyzed par , 
 returning much of the hparp-1 to its native size by coomassie ( figure 2a ) and removing p - labeled par , as shown 
 in the autoradiograph ( figure 2b ) .
these results 
 suggest that the standard alkaline conditions in phosphoproteomic 
 elution protocols result in the loss of parylation , while the neutral 
 phosphate buffer preserves the adpr - protein bond and should be a safe 
 method to elute phospho(ribosyl)ated peptides from phospho - affinity 
 matrices .
( a ) coomassie staining shows that parylated hparp-1 returns 
 to its unmodified size upon treatment with ammonia for 5 min , while 
 neutral phosphate retains the parylation state as well as the control 
 buffer ( automodification buffer ) .
( b ) p - labeled par shows 
 that the loss of par is correlated to the return of hparp-1 to its 
 native size .
bsa ( bovine serum albumin ) was included as a carrier 
 for sample cleanup by protein precipitation , which was the method 
 applied to immediately quench the chemical exposure .
next , we 
 explored whether phospho(ribosyl)ated peptides can be enriched from 
 cellular complex mixtures using phosphoenrichment matrices .
svp - treated 
 hparp-1 was mixed with hela cell lysate , which was silac labeled in 
culture medium 
 containing c6,n2-lysine 
 and c6,n4-arginine 
 ( supplementary figure 1 in the supporting information ) . because we expect the human hparp-1 spectra to be derived from 
 svp - treated , unlabeled  light 
hparp-1 samples , we can 
 verify the source of the peptide by silac state . as a positive phosphoenrichment 
 control ,
peptides from known phosphoprotein standards , bovine caseins , 
 were also added to the whole cell lysate background .
the complex peptide 
 mixture was subjected to three commercially available phosphoenrichment 
 matrices : ( 1 ) sigma phos - select iron affinity gel ( ps ) , ( 2 ) gl science 
 titansphere phos - tio2 tips ( gl ) , and ( 3 ) glysci phosphopeptide 
 nutip using zirchrom titanium dioxide beads ( zc ) . in each case
, peptides 
 were eluted with 0.5 m potassium phosphate buffer at ph 7.0 to preserve 
 the labile bond between phospho(ribose ) and acidic amino acids .
mass 
 spectrometry data were collected on an orbitrap , and fragmentation 
 was induced by both collision - induced dissociation ( cid ) and higher - energy 
 c - trap dissociation ( hcd ) .
overall , our complex background consisted 
 of 44  655 peptides from 2148 proteins and included 3421 endogenous 
 phosphopeptides .
( see supplementary tables 24 in the supporting information . ) out of this background 
 we identified 47 unique phosphopeptides from the spiked - in phosphoprotein 
 standards ( bovine caseins ) using all enrichment techniques ( supplementary 
 table 1 in the supporting information , 
 figure 3a , and supplementary figure 2a , e in 
 the supporting information ) . while phos - select 
 contributed the most unique peptide identifications ( 36% ) , both gl 
 sciences and zirchrom found peptides that would not have otherwise 
 been identified ( 2 and 13% , respectively ) .
this stands in contrast 
 with the 29 unique hparp-1 phospho(ribosyl)ated peptides , of which 
 nearly 60% were found exclusively through enrichment by phos - select 
 ( supplementary table 1 in the supporting information , figure 3b , and supplementary figure 2b , f 
 in the supporting information ) , and only 
 a single peptide ( 3% ) was found solely by an alternative enrichment 
 ( zirchrom ) .
further assessment of the phos - select enrichment profile 
 reveals that the 39 unique phosphopeptides and 28 unique phospho(ribose ) 
 peptides found in the phos - select eluate entirely overlapped with 
 the small number of peptides , which were found in the respective input 
 and flowthrough analyses ( figure 3c , d and supplementary 
 figure 2c , d , g , h in the supporting information ) .
imac ( phos - select , ps ) was compared with moac ( both zirchrom , zc and 
 gl sciences , gl ) for enrichment of phosphopeptides ( from bovine casein ) 
 and phospho(ribose ) peptides ( from hparp-1 ) out of hela whole cell 
 lysate background .
( a , b ) unique phosphorylated ( a ) and phospho(ribosyl)ated 
 ( b ) peptides identified in eluates from the three methods .
( c , d ) unique 
 phosphorylated ( c ) and phospho(ribosyl)ated ( d ) peptides identified 
 in the unenriched ( input ) , elution , and flowthrough from the imac 
 method . to determine whether the protocol 
 proposed here is as robust for 
 phospho(ribosyl)ated peptides as phosphopeptides
, we performed a serial 
 enrichment that included re - enriching the flowthrough sample multiple 
 times to quantify the depletion of these two classes of target peptides 
 ( figure 4 ) .
automodified hparp-1 was again 
 used as the par standard ; however , this time the parylated hparp-1 
 was denatured in 8 m urea , reduced , and alkylated prior to being added 
 to the whole cell lysate background ( figure 4a ) .
this denaturation step served to completely inactivate hparp-1 
 ( see supplementary figure 3 in the supporting 
 information ) , thus allowing us to perform svp digestion of 
 the whole cell lysate and the parylated standard in the same mixture . 

furthermore , the his - tag on hparp-1 allowed us to isolate a portion 
 of the standard back out of the mixture both before and after svp 
 digestion ; these samples served as a quality - control step as the loss 
 of par and the formation of phospho(ribose ) could be monitored by 
 sds - page and western blot .
both classes of peptides 
 are depleted from the background population at similar rates ( as opposed 
 to phosphopeptides being enriched preferentially prior to phospho(ribosyl)ated 
 peptides ) , indicating that the imac method proposed is truly a dual 
 enrichment of both phospho- and phospho(ribosyl)ated peptides .
it 
 should be noted that the peptides identified in this study include 
 both those from the hparp-1 standard as well as the endogenous phospho- 
 and phospho(ribosyl)ation sites from the mnng - treated murine parg 
 knockout cells used to generate the heavy - labeled complex background . 
 for a complete list of endogenous phospho- and
his - tagged , automodified parp-1 was 
 denatured in 8 m urea and spiked into heavy - labeled whole cell lysate 
 from mnng - treated murine parg knockout cells before being treated 
 by svp and then digested to peptides and enriched three times in a 
 row on imac beads ( a ) .
samples were taken before and after svp treatment , 
 and the his - tagged parp-1 was separated from the whole cell lysate 
 by nickel - chelated agarose beads , allowing visualization of the total 
 protein ( b ) , parylated his - parp-1 ( c ) , and phospho(ribosyl)ated his - parp-1 
 ( d ) .
ms / ms analysis of the serial enrichments showed that the endogenous 
 phospho - peptides and the phospho(ribosyl)ated parp-1 peptides were 
 depleted from the complex mixture at similar rates ( e ) . among 
 the phospho(ribosyl)ated peptides identified from the hparp-1 standard , 
 20 unique sites were modified .
many of these sites were outside of 
 the automodification / brct domains that are known to be heavily parylated ( table 1 ) , and in fact , 
 over one - third of the sites identified ( 7/20 ) are in the second zinc 
 finger , which is not strictly required for parp-1 activation . of the 20 potential parylation sites , 1 arginine , 
 3 lysine , 4 aspartate , and 12 glutamate residues were identified . 

while the basic sites may seem surprising we emphasize that the inherent 
 nadase activity of parp-1 has the potential 
 to create free adpr , a molecule that can spontaneously modify basic 
 sites independent of parp-1 s conjugation activity . because this nonenzymatic mechanism of adp - ribosylation 
 is still under investigation
, we believe the ability of this method 
 to identify the presence of adpr on both basic and acidic modifications 
 will prove highly useful in elucidating methods of adpr modification 
 and automodification .
a total of 20 automodification 
 sites were identified on the parp-1 standard used for assessing phosphoenrichment 
 techniques .
those that were identified by zhang et al . are known to be 
 endogenous parylation sites . zf1 = zinc finger 1 , zf2 = zinc finger 
 2 , brct = brca1 c - terminus , and wgr = tryptophan , glycine , arginine - rich . among the 20 hparp-1 automodification 
 sites
identified , 10 were 
 independently verified as endogenous sites in dna damaged cells in 
 a recent analysis . while most peptides 
 presented with a single phospho(ribose ) , there were three examples 
 of doubly phospho(ribosyl)ated peptides that demonstrated the ability 
 of hparp-1 to place these large , negatively charged polymers within 
 close proximity of each other ( supplementary spectra in the supporting information ) .
a notable example of 
 this is the dual modification of e488 and e491 parp-1 automodification 
 sites , which have been independently verified by a number of groups , 
 including zhang et al . , who identified them as endogenous adp - ribosylation 
 sites ( table 1 ) .
here we have shown the fragmentation 
 patterns of the unmodified , singly modified and doubly modified forms 
 of this peptide by both cid ( figure 5 ) and 
 hcd ( supplementary figure 4 in the supporting 
 information ) , indicating the shift in molecular weight corresponding 
 to a single ( circle ) or a double ( square ) phospho(ribose ) group .
the 
 doubly modified peptide also demonstrates the potential for neutral 
 loss of phosphoric acid ( h3po4 97.98 da ) and 
 phospho(ribose ) ( c5h9po7 , 212.01 
 da ) from the parent ion upon fragmentation ( figure 5 , supplementary figure 5 in the supporting 
 information ) ; these neutral losses were observed in 73% ( 16/22 ) 
 of the spectra annotated for validation of the parp-1 automodification 
 sites ( supplementary spectra in the supporting 
 information ) , most often showing up in the presence of the 
 modified form , indicating that neutral loss was not complete .
considering 
 how common these neutral losses are , the authors advise including 
 them in mass spectrometry search criteria .
e488 and e491 are previously 
 characterized parp-1 automodification sites , shown here in panels 
 b and c , respectively , as compared with the unmodified form of the 
 peptide shown in panel a. the doubly modified peptide ( d ) contains 
 diagnostic fragments that carry one phosphoribose group ( 212.01 da , 
 circles ) as well as those carrying two phosphoribose groups ( 424.02 
 da , squares ) .
our analysis identified 
 three lysine modifications : two novel and 
 one previously reported in a 2009 mutagenesis screen ( table 1 ) .
two of these were found 
 at the c - terminus of the peptide , suggesting that the phospho(ribosyl)ated 
 residue did not prevent proteolytic cleavage at the modified lysine , 
 in our case by a combination of lysc and trypsin . to confidently assign 
 the novel parylation site k486 , its cid fragmentation pattern was 
 compared with an unmodified version of the same peptide , revealing 
 a b - ion series that was unmodified in both spectra and a y - ion series 
 that contained the 212.01 da shift indicative of a phospho(ribose ) 
 addition to every y - ion fragment ( figure 6 ) . 

the extensive b- and y - ion series provide strong evidence of the phospho(ribose ) 
 modification on the peptide c - terminal lysine , demonstrating ( 1 ) the 
 availability of phospho(ribosyl)ated lysines for protease cleavage 
 and ( 2 ) the ability of phos - select to enrich phospho(ribosyl)ated 
 lysines .
k486 , a novel 
 parp-1 parylation site identified in our analysis , is shown here at 
 the peptide c - terminus ( b ) .
this fragmentation pattern is compared 
 with that of the unmodified form ( a ) showing the characteristic 212.01 
 da shift present in the entire y - series but absent from the b - series , 
 validating the localization of phospho(ribose ) . to establish 
 a pipeline for identifying endogenous sites of mono- 
 and poly(adp - ribosyl)ation ,
hela cells were silac - labeled and then 
 treated with the dna damaging agent mnng to induce parylation before 
 being subjected to an affinity pull - down by the mono- and poly(adp - ribose ) 
 binding macrodomain from af1521 .
adp - ribosylated proteins were then denatured before being 
 treated with svp and then digested with a mixture of the proteases 
 lysc and trypsin .
these peptide mixtures were then split in half and 
 either enriched over a charged or a stripped imac resin with the elution 
 from the stripped resin serving as a nonspecific background control 
 for the eluted peptides that had come off of the charged imac resin . 

( see figure 7a . ) because both forward- and 
 reverse - labeling patterns were used , the strongest hits from the database 
 showed up in both populations , as demonstrated in figure 7b , c . a representative spectrum for phosphoribosylated 
 r4 from serine / arginine - rich splicing factor 2 ( srsf2 ) is shown with 
 its parent ion in figure 7b , d .
notably , the 
 pipeline described in figure 7a was performed 
 in parallel on an mnng - treated trophoblast stem cell line from a parg 
 knockout mouse model , producing 22 unique 
 endogenous phospho(ribosyl)ated peptides , two of which ( containing 
 r4 from srsf2 and r199 from heterogeneous nuclear ribonucleoprotein 
 u , hnrnpu ) overlapped with those found from the hela preparation . 

all of the phospho(ribosyl)ated peptides identified from these 
 samples were found in the imac enriched fractions , indicating that 
 macrodomain enrichment followed by svp digestion was not sufficient 
 for site identification .
to 
 identify adp - ribosylation 
 sites from whole cells , we mnng - treated hela cells that were either 
 heavy ( k8r10 ) or light ( k0r0 ) labeled , affinity - enriched adp - ribosylated proteins , treated these 
 proteins with svp to yield phosphoribose , and then digested these 
 proteins to a peptide mixture that would then be enriched by either 
 charged or stripped imac beads ( a ) . stripped imac beads from each 
 population would serve as a background control for the reverse labeled 
 peptides enriched over a charged matrix .
this example shows the ms 
 ( b , c ) spectra of both the heavy and light forms of r4 from serine / arginine - rich 
 splicing factor 2 ( srsf2 ) as well as the annotated ms / ms of the light 
 form ( d ) .
serine ( gray ) carries a protein n - terminal acetylation , 
 arginine ( red ) carries phospho(ribose ) . to determine whether the macrodomain enrichment was necessary 
 for 
 site identification , we did the same analysis with the adpr affinity 
 purification omitted , again utilizing both the human wild - type and 
 murine parg knockout cell lines previously described .
twenty - two unique 
 phospho(ribosyl)ated peptides were identified from these preparations , 
 including the same hnrnpu peptide containing r199 found following 
 macrodomain enrichment ( it was again found in both cell lines ) , showing 
 that the macrodomain enrichment is not only insufficient on its own 
 for site identification but also that it is not necessary
. furthermore , 
 a comparison of the macrodomain enriched versus unenriched data sets 
 revealed a bias in the amino acids , which served as attachment sites 
 for phospho(ribose ) ; the macrodomain enrichment appears to have shifted 
 the profile of adp - ribosylated amino acids away from glutamic acid 
 residues ( figure 8 , source data can be found 
 in the supplementary text and supplementary table 5 in the supporting information ) .
this shift indicates 
 that the macrodomain is either selecting against adp - ribosylated glutamic 
 acid in favor of other amino acid attachment sites or that it is actually 
 removing the adpr attachment from glutamic acids .
the latter hypothesis 
 lines up with recently published work showing that the macrodomain 
 of af1521 possesses adp - ribosylhydrolase activity 
 and suggests that this activity may be targeted toward glutamic acid 
 sites of adp - ribosylation .
unique phospho(ribosyl)ated peptides identified from whole 
 cells , as detailed in supplementary table 5 in the supporting information , show a shift in the profile of amino 
 acids carrying phospho(ribose ) from both human wild - type ( a ) and murine 
 parg knockout cells ( b ) .
the expanding relevance of parylation in 
 cellular processes has 
 led researchers to look beyond the canonical role of dna repair when 
 considering the consequences of altered parylation levels . to this end ,
the most powerful tool for studying 
 global changes in post - translational modifications continues to be 
 systematic analyses of proteomes by mass spectrometry .
unfortunately 
 the widespread use of proteomics and mass spectrometry has not yet 
 been established in the field of parylation due to challenges relating 
 both to the modification itself , which may be labile , large , and highly 
 charged , and to the low levels of parylation that exist below the 
 threshold of most analytical techniques . in response ,
enrichment techniques 
 have been developed that have allowed researchers to study the parylated 
 proteome with the caveat that the identified proteins are either par 
 acceptors or par binders ; due to the lack of site identification in 
 these studies , verification of which class these proteins belong to 
 is both tedious and , in some cases , impossible . recently
, a study has demonstrated the feasibility of identifying 
 mono- and poly(adp - ribosyl)ation sites in a large proteomic screen 
 that combines enrichment of adp - ribosylated substrates by boronate 
 chromatography with the removal of adpr from substrates by hydroxylamine ; 
 this chemical treatment allows for subsequent identification of acidic 
 adp - ribosylation sites by the diagnostic 15.01 da hydroxamic acid 
 derivative left behind .
the limitation 
 in this study was that adp - ribosylated lysine and arginine could not 
 be detected as only acidic residues were left with the hydroxamic 
 acid tag .
in contrast , our pipeline can identify adp - ribosylation 
 attachment sites on both acidic and basic residues ; it should also 
 be noted that this universality allows for the discovery of novel 
 amino acid attachment sites for adpr beyond these acids and bases , 
 the existence of which has not been ruled out .
we believe our proposed 
 method of enriching and identifying adp - ribosylation sites addresses 
 the need for a pipeline that is both global and definitive in identifying 
 adpr acceptors at the protein and amino acid levels .
the phosphoenrichment 
 methods applied in this study have gained 
 popularity in the phosphoproteomic field due to their high specificity 
 and compatibility with both maldi and esi  lc  ms .
moac 
 has proven to enrich phosphopeptides more specifically than its predecessor , 
 imac , likely due to the tighter binding of phosphate to the tio2 microspheres ( titanspheres ) as compared with the chelated 
 iron used by phos - select imac resin .
this 
 tight binding , however , may explain the lack of phospho- and phospho(ribose ) 
 peptides found in the eluates from the tio2 resins used 
 here ( gl sciences and zirchrom ) , which have an optimal elution ph 
 between 9.2 and 9.4 .
imac elution is 
 much more sensitive to competitive phosphate levels than it is to 
 ph and does not have an optimal elution ph .
we have demonstrated the stability of adpr protein attachment sites 
 in neutral phosphate buffer as compared with basic nh4oh 
 and have restricted our elution conditions to ensure retention of 
 phospho(ribose ) on target peptides throughout the enrichment .
this 
 consideration may have favored the lower - affinity phosphoenrichment 
 matrix , allowing for a single enrichment protocol capable of enriching 
 both phospho- and phospho(ribose ) peptides , perhaps at the expense 
 of tightly bound phosphopeptides left on the tio2 matrices . 
 for thorough phosphopeptide analysis
, it may be prudent to perform 
 a parallel enrichment with optimal ( i.e. , basic ) elution conditions 
 from a tio2 matrix .
while validating the presence 
 of our phospho(ribosyl)ated protein 
 sample , we discovered that the phosphoprotein sds - page gel stain , 
 pro - q diamond , can act as an indicator of phospho(ribose)-modified 
 proteins . while we did not do any in - gel digests , the compatibility 
 of pro - q diamond with downstream lc 
ms analysis suggests that isolation and identification of 
 phopho(ribosyl)ated proteins as well as their par acceptor sites may 
 be possible for researchers who wish to analyze changes in sds - page 
 profiles .
we believe this data - dependent approach would greatly complement 
 the global analysis already offered by the phospho(ribose ) adp - ribosylation 
 tag .
optimization of our phosphoenrichment protocol presented 
 us with 
 a database of spectra identifying phospho(ribosyl)ated peptides from 
 automodified hparp-1 , ultimately yielding 20 modified sites , eight 
 of which are being reported for the first time .
these spectra afforded 
 us the opportunity to characterize phospho(ribosyl)ated peptides ( and 
 by extrapolation , adp - ribosylation sites ) with regard to their identification 
 by cid- and hcd - assisted lc 
first , we determined that 
 multiple parylation sites may exist within the same peptide , suggesting 
 that hparp-1 is capable of placing these large , highly charged polymers 
 within an amino acid of each other ( as in the hparp-1 automodified 
 peptide gfsllate*d*k ; see supplementary spectra in the supporting information ) . the steric hindrance 
 and charge - repulsion associated with neighboring parylation sites 
 may require a high level of flexibility from the protein , poly(adp - ribose ) , 
 or both .
second , it is worth noting that we identified two lysine 
 hparp-1 automodification sites at the c - terminal end of their respective 
 peptides , indicating that these modified lysines were available for 
 proteolytic digestion .
( see supplementary table 2 in the supporting information . ) finally , fragmention 
 by hcd and cid revealed the potential of phospho(ribose ) to be partially 
 or fully lost in the form of a phosphoric acid or phosphoribose , respectively . 

this loss is not complete as the fragments portraying 
 the neutral loss are often accompanied by otherwise - identical fragments 
 that have maintained the full modification . in the future
, these neutral 
 loss fragments may serve as diagnostic indicators of peptide phospho(ribosyl)ation 
 state .
recognition of these attributes will aid in the analysis of 
 large , phospho(ribosyl)ated proteomes , which may present these characteristics 
 that would allow them to be ignored by erroneous search parameters .
while demonstrating the application of this method to identify 
 adp - ribosylation sites from whole cells , we validated several known 
 sites of adp - ribosylation recently identified by a complementary mass 
 spectrometry approach as well as a host 
 of novel sites on both novel and known acceptors of mono- or poly(adp - ribose ) . 

one of our most interesting hits lies on k350 of heterogeneous 
 nuclear ribonucleoprotein a1 ( hnrnpa1 ) , a protein that was first shown 
 to be poly(adp - ribosyl)ated in whole cells in 1982 and 12 years later 
 was shown to be one of the two major acceptors of adpr in hela cells .
more recently , parylation of hnrnpa1 has been 
 shown to affect splicing , stem - cell maintenance , and oocyte localization 
 in drosophila , suggesting an interesting role for mammalian hnrnpa1 
 parylation . while there have been several 
 proteomic studies that have identified hnrnpa1 in poly or mono(adp - ribosyl)ation 
 purification schemes , this finding is 
 the first indication of the site of parylation on hnrnpa1 ( spectrum 
 annotated in supplementary figure 6 in the supporting 
 information ) . in summary , we have proposed and demonstrated 
 the feasibility of 
 a global , unbiased approach for characterizing the mono- and poly(adp - ribosyl)ated 
 proteome .
our technique , based on the digestion of adpr down to its 
 phospho(ribose ) attachment site , allows for enrichment at the peptide 
 level of both acidic and basic adpr acceptor sites .
furthermore , we 
 have shown that our method allows researchers to find sites of adp - ribosylation 
 without having to knock down adpr hydrolases or perform an enrichment 
 of the adp - ribosylated proteome , steps that may otherwise introduce 
 bias .
finally , this approach presents a unique opportunity to study 
 the changes in the adp - ribosylated proteome alongside the coenriched 
 phosphoproteome .
it is our hope that the accessibility of the techniques 
 employed in this enrichment pipeline will allow researchers to characterize 
 global adp - ribosylation at the level of the amino acid , ultimately 
 resulting in a greater understanding of both mono- and poly(adp - ribose ) 
 function and regulation from the bottom up .
 poly(adp - ribose ) , 
 or par , is a cellular polymer implicated in dna / rna 
 metabolism , cell death , and cellular stress response via its role 
 as a post - translational modification , signaling molecule , and scaffolding 
 element . 
 par is synthesized by a family of proteins known as poly(adp - ribose ) 
 polymerases , or parps , which attach par polymers to various amino 
 acids of substrate proteins . 
 the nature of these polymers ( large , 
 charged , heterogeneous , base - labile ) has made these attachment sites 
 difficult to study by mass spectrometry . here we propose a new pipeline 
 that allows for the identification of mono(adp - ribosyl)ation and poly(adp - ribosyl)ation 
 sites via the enzymatic product of phosphodiesterase - treated adp - ribose , 
 or phospho(ribose ) . 
 the power of this method lies in the enrichment 
 potential of phospho(ribose ) , which we show to be enriched by phosphoproteomic 
 techniques when a neutral buffer , which allows for retention of the 
 base - labile attachment site , is used for elution . through the identification 
 of parp-1 in vitro automodification sites as well as endogenous adp - ribosylation 
 sites from whole cells 
 , we have shown that adp - ribose can exist on 
 adjacent amino acid residues as well as both lysine and arginine in 
 addition to known acidic modification sites . 
 the universality of this 
 technique has allowed us to show that enrichment of adp - ribosylated 
 proteins by macrodomain leads to a bias against adp - ribose modifications 
 conjugated to glutamic acids , suggesting that the macrodomain is either 
 removing or selecting against these distinct protein attachments . 
 
 ultimately , the enrichment pipeline presented here offers a universal 
 approach for characterizing the mono- and poly(adp - ribosyl)ated proteome . 
Introduction
Materials and Methods
Results
Discussion