nitric oxide ( no ) is a pleiotropic signaling 
 molecule produced 
 endogenously by no synthases in diverse biological systems .
s - nitrosylation is the addition of a no group to a specific 
 cysteine ( cys ) residue of a protein to form a s - nitrosothiol . 

this prototypic , redox - based post - translational protein modification 
 process has been increasingly recognized as a key cellular and molecular 
 mechanism for no signaling and plays an important role in physiological 
 conditions and in diseases such as diabetes , asthma , heart failure , 
 cancer , and neurodegenerative disorders.s - nitrosylation 
 of critical cys residues on specific proteins regulates their activities 
 and has been demonstrated in protein misfolding resulting in interference 
 of normal cellular functions . in 
 order to investigate mechanisms underlying no signaling , a number 
 of methods have been established for identifying s - nitrosylated proteins ( sno - proteins ) and mapping their s - nitrosylated cysteine ( sno - cys ) sites . the biotin switch technique 
 ( bst ) developed by jaffrey et al .
it involves first blocking free thiols with a sulfhydryl - reactive 
 reagent , such as s - methyl methanethiosulfonate ( mmts ) , 
 then selectively reducing the s - nitrosothiols with 
 ascorbate , and labeling with n-[6-(biotinamido)hexyl]-3-(2-pyridyldithio ) 
 propionamide ( biotin - hpdp ) .
the biotinylated sno - proteins can be enriched 
 via avidin - agarose affinity capture , and then proteins of interest 
 are detected by immunoblotting . despite its popularity , this method 
 is relatively low - throughput , and it is difficult to locate the modification 
 sites .
additionally , the biotin affinity enrichment may introduce 
 false positive signals due to the existence of endogenous biotin - like 
 molecules . to specifically assess sno - cys modification status and 
 increase the throughput of detection , several mass spectrometry ( ms)-based 
 methods have been developed by modifying the bst protocol , including 
 sno - cys site identification ( snosid ) , s - nitrosothiol 
 resin - assisted capture ( sno - rac ) , s - alkylating labeling 
 strategy , and cystmt switch assay . however ,
none of these modified bst methods 
 are able to achieve all of the following features desired for comprehensive 
 characterization of sno - cys modification : a simple , robust workflow 
 for sno - protein identification , an irreversible thiol linkage for 
 effective peptide digestion and for unambiguous sno - cys mapping , as 
 well as sample multiplexing for increased throughput and relative 
 quantification of sno - proteins under various conditions . in 
 this study , we present a novel ms - based modified bst , the iodotmt 
 switch assay ( isa ) , using cys thiol - reactive iodoacetyl tandem mass 
 tag ( iodotmt ) sixplex reagents to identify and quantify protein s - nitrosylation . with a simple workflow , isa accomplishes 
 irreversible labeling of sno - cys sites , s - nitrosylated 
 peptide ( sno - peptide ) enrichment using high affinity anti - tmt chromatography 
 with competitive elution , and multiplex quantification of protein s - nitrosylation via six unique tmt mass reporter ions .
notably , 
 this workflow is capable of detection and quantification of sno - cys 
 modifications under physiological and pathological conditions . in 
 this study
, we applied this approach to the investigation of protein s - nitrosylation in a neuroinflammation modelendotoxin 
 lipopolysaccharide ( lps)-stimulated immortalized murine bv-2 microglial 
 cells  representing a convergent nitrosative stress and neuroinflammatory 
 response in the pathogenesis of age - associated neurodegenerative diseases .
we further evaluated the effects of s - allyl cysteine 
 ( sac ) , an active nutritional compound of aged garlic extract ( age ) , 
 on lps - stimulated s - nitrosylation in bv-2 cells .
the iodotmtsixplex reagents , anti - tmt antibody , 
 immobilized anti - tmt antibody resin , bicinchoninic acid ( bca ) protein 
 assay kit , c18 tips , and primary antibodies for protein validation 
 were obtained from thermo fisher scientific ( rockford , il ) .
sep - pak tc18 1 cc vac cartridges were purchased from waters ( dublin , 
 ireland ) .
amersham ecl prime western blotting detection reagent was 
 purchased from ge healthcare ( buckinghamshire , uk ) .
trypsin ( modified , 
 sequencing grade ) was obtained from promega ( madison , wi ) .
dulbecco s 
 modified eagle s medium ( dmem ) , penicillin - streptomycin , and l - glutamine were obtained from gibco ( grand island , ny ) .
all 
 other reagents including sac ( a garlic active component ) were from 
 sigma - aldrich ( st . louis , mo ) .
the purity and stability of sac were 
 validated prior to use through the nutrition core of the center for 
 botanical interaction studies at the university of missouri .
the immortalized murine 
 bv-2 microglial cells were cultured in dmem containing 5% ( v / v ) heat - inactivated 
 fbs , 25 u / ml penicillin , and 25 g / ml streptomycin at 37 c 
 in a saturated humidity atmosphere containing 95% ( v / v ) air and 5% 
 ( v / v ) co2 .
cells were plated at a density of 1  10/ml medium in 35 mm or 100 mm plates and grown to 70% confluence 
 prior to testing .
bv-2 cells were starved for 4 h in fbs - free dmem 
 ( without phenol red ) and then exposed to 100 ng / ml lps for 16 h in 
 the presence or absence of 5 mm sac or 0.5 mm n--nitro - l - arginine methyl ester hydrochloride ( l - name ) .
after lps stimulation , conditioned medium was 
 collected and mixed with an equal volume of griess reagent [ 1% ( w / v ) 
 sulfanilamide and 0.1% ( w / v ) n-(1-naphthyl ) ethylenediamide 
 in 5% ( v / v ) phosphoric acid ] .
after a 10 min incubation at room temperature 
 ( rt ) , absorbance was read at 543 nm using a biotek synergy-4 microplate 
 reader ( biotek instruments , inc . ,
a series of sodium nitrite dilutions ( 0100 m ) was used 
 to generate a nitrite standard reference curve .
after treatment , 0.5 mg / ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2h - tetrazolium bromide ( mtt ) was added to the cell culture . 

after incubation at 37 c for 4 h , the formed formazan crystals 
 were collected and dissolved in dimethyl sulfoxide ( dmso ) .
the absorbance 
 at 540 nm was read using the synergy-4 microplate reader . for in vitro s - nitrosylation , 
 400 g of bv-2 cell lysates in hents buffer 
 [ 250 mm hepes - naoh , ph 7.4 , 1 mm edta , 0.1 mm neocuproine , 1% ( v / v ) 
 triton x-100 , 0.1% ( w / v ) sds , 1% ( v / v ) protease inhibitor cocktail ] 
 were exposed to a final concentration of 200 m s - nitrosocysteine ( snoc ) for 30 min at rt in the dark . in 
 vivo
treated bv-2 cells were rinsed twice in cold phosphate - buffered 
 saline followed by lysis in hents buffer .
free sulfhydryl groups in the protein sample were first 
 blocked with 20 mm mmts for 30 min at 50 c with frequent vortexing 
 in hen ( 250 mm hepes - naoh , ph 7.4 , 1 mm edta , 0.1 mm neocuproine ) 
 buffer containing 2.5% ( w / v ) sds . to remove excess mmts , proteins 

were precipitated using 4 volume of cold acetone for 30 min 
 at 20 c .
proteins were then redissolved in hen buffer 
 containing 1% ( w / v ) sds and labeled with 1 mm iodotmt reagent in the 
 presence of 5 mm sodium ascorbate for 2 h at rt .
for ms analysis , after 
 acetone precipitation , iodotmt - labeled proteins were redissolved with 
 tris - hcl , ph 8.0 , containing 0.1% ( w / v ) sds .
six different protein 
 samples individually labeled by iodotmtsixplex reagents were combined 
 before tryptic digestion .
the iodotmt - labeled proteins were reduced 
 with 10 mm dithiothreitol 
 ( dtt ) at 55 c for 1 h and then alkylated with 25 mm iodoacetamide 
 at 37 c for 1 h in the dark .
after acetone precipitation , the 
 protein pellet was resuspended with 50 mm ammonium bicarbonate containing 
 20 ng/l trypsin and digested overnight at 37 c .
undigested 
 proteins were removed by centrifuge at 2500 g , rt 
 for 10 min .
the peptides were then desalted with 50 mg waters sep - pak 
 tc18 columns and eluted with 50% ( v / v ) acetonitrile ( acn ) and 0.1% 
 ( v / v ) trifluoroacetic acid ( tfa ) .
a small portion of this unfractionated 
 sample was retained for future analysis of the unenriched sample . for peptide enrichment ,
the above samples were lyophilized and redissolved 
 in 600 l of tbs buffer ( 25 mm tris - hcl , ph 7.5 , 0.15 m nacl ) . 

typically , 100 l of settled immobilized anti - tmt antibody resin 
 is employed for enrichment of every 1 mg of s - nitrosylated 
 sample . in our experiments ,
the peptide sample was incubated with 
 200 l of settled anti - tmt antibody resin overnight at 4 c 
 with end - over - end rocking .
after the supernatant was removed , the 
 resin was washed three times with one column volume of tbs and three 
 times with one column volume of milli - q water .
peptides were finally 
 eluted with four column volumes of elution buffer [ 10 mm cis-2,6-dimethylpiperidine 
 ( 2,6-dmpp)/500 mm tetraethylammonium bicarbonate ( teab ) , ph 8.5 ] . a easy - nlc 1000 hplc system and easyspray 
 source ( thermo scientific ) with easy - spray pepmap rslc c18 25 cm  
 75 m i d column ( thermo scientific )
were used to separate peptides 
 with a 525% ( v / v ) acn gradient in 0.1% ( v / v ) formic acid over 
 120 min at a flow rate of 300 nl / min .
samples were analyzed on orbitrap 
 elite and ltq orbitrap xl mass spectrometers ( thermo scientific ) using 
 top 15 fourier transform ( ft ) ms / ms with higher - energy collision dissociation 
 ( hcd ) or top 3/3 ion - trap collision - induced dissociation ( cid)/ft 
 hcd experiments .
proteome discoverer software version 1.4 ( thermo 
 scientific ) was used to search ms / ms spectra against the swiss - prot 
 mouse database using the sequest ht or mascot 2.3 search engines . 

dynamic modifications included carbamidomethylation ( c ) , iodotmtsixplex 
 ( c ) , and methionine oxidation .
resulting peptide hits were filtered 
 for a maximum 5% false discovery rate using the percolator .
the iodotmtsixplex 
 quantification method within proteome discoverer software was used 
 to calculate the reporter ratios with a mass tolerance 10 ppm .
anti - tmt antibody affinity 
 was determined using a tmt - derivatized cm5 chip on a biacore 3000 
 instrument ( ge healthcare ) . for competitive elution buffer scouting
, 
 anti - tmt antibody was first bound to tmt - derivatized surface followed 
 by a timed injection of tmt analogue compounds . for sds - page ,
4 sds 
 sample buffer with 1% ( v / v ) 2-mercaptoethanol was added to the protein 
 samples .
proteins were then resolved on 10% , 1 mm sds - page gels . for 
 immunoblotting
the membrane was first blocked in phosphate - buffered saline 
 plus 0.1% ( v / v ) tween-20 ( pbst ) containing 5% ( w / v ) nonfat milk at 
 rt for 1 h followed by incubation with primary antibody in pbst at 
 4 c overnight . after being washed three times with pbst , the 
 membrane was incubated with secondary antibody ( anti - mouse igg - peroxidase 
 antibody produced in goat or anti - rabbit igg - peroxidase antibody produced 
 in goat , sigma ) in pbst at rt for 1 h. immuno - reactive bands were 
 visualized using amersham ecl prime western blotting detection reagent 
 and detected with las-4000 imaging system ( fujifilm ) .
degree of freedom and z score for 
 each iodotmt modification site was calculated 
 by a special student s t - test , known as welch s t - test .
we further used a t - distribution implemented in the 
 r statistical package to calculate the p - value based 
 on degree of freedom and z score .
s - nitrosylation level change of a sno - cys was considered as significant 
 if its fold change is greater than 1.3 and p - value 
 is less than 0.05 .
motif consensus sequences 
 for protein s - nitrosylation was analyzed by the motif - x 
 algorithm .
sno - proteins with significant s - nitrosylation level changes were subjected to functional 
 annotation 
 by ingenuity pathway analysis ( ipa ; http://www.ingenuity.com ) and the in - house multicom - pdcn software .
iodotmtsixplex reagents are commercially available and were used 
 in the study to determine protein s - nitrosylation . 

each isobaric iodotmtsixplex reagent within a set has the same nominal 
 mass and consists of a thiol - reactive iodoacetyl group , a ms - neutral 
 spacer arm , and a ms / ms reporter .
the chemical structure of iodotmt-126 
 ( one of the sixplex reagents ; 126 refers to the mass of the reporter 
 ion ) is shown in figure 1a . during ms /
ms analysis 
 to derive fragment ions and sequence information , the isobaric mass 
 tags are cleaved generating reporter ions with unique m / z of 126131 . in isa , after blocking free 
 thiols in the proteins with mmts , an iodotmt reagent is used to irreversibly 
 label nascent thiols generated by the selective reduction of sno - cys 
 bonds by ascorbate ( figure 1b ) .
the irreversibility 
 of iodotmt labeling was validated under our tested conditions ( supplemental figure s1 , supporting information ) . to test the specificity of iodotmt labeling ,
bv-2 cell lysates 
 were s - nitrosylated in vitro by 
 exposure to a physiological no donor snoc ( 200 m ) and followed 
 by labeling with iodotmt-126 .
as shown in figure 1c , iodotmt - labeled samples showed much stronger signals than 
 control conditions without snoc and/or ascorbate on the anti - tmt immunoblot . 
 to test the sensitivity of the iodotmt labeling assay ,
bv-2 cell lysates 
 were treated with different concentrations ( 10 , 50 , 100 , 200 m ) 
 of snoc .
after switch labeling with iodotmt-126 , sno - protein signals 
 in snoc - treated samples exhibited a dose - dependent increase compared 
 to the untreated control sample ( figure 1d ) . 

these results suggested that iodotmt switch labeling is feasible and 
 reliable for detecting sno - proteins and is sensitive to as low as 
 10 m snoc treatment .
( a ) the chemical structure of iodotmt-126 reagent . 
 the iodotmtsixplex reagents including iodotmt-126 illustrated here 
 consist of a cys thiol - reactive iodoacetyl group , a mass normalizing 
 spacer arm , and a mass reporter .
( b ) reaction scheme for the labeling of sno - protein by 
 iodotmt reagents .
sno - cys residues 
 are then selectively released by ascorbate and labeled by thiol - reactive 
 iodotmt reagents .
s - nitrosylated by snoc ( 200 m ) were switch - labeled by iodotmt-126 . 

this sample exhibited strong signals on the anti - tmt immunoblot , whereas 
 controls omitting snoc and/or ascorbate displayed much lower signal 
 levels .
( d ) bv-2 cell lysates exposed to snoc ( 0 , 10 , 50 , 100 , 200 
 m ) were switch - labeled by iodotmt-126 .
as the concentration 
 of snoc increased , more sno - proteins were detected on the anti - tmt 
 immunoblot .
the protocol allows both sno - protein 
 quantification and sno - cys 
 site - mapping .
furthermore , up to six different protein samples can 
 be simultaneously analyzed with iodotmtsixplex reagents , iodotmt126131 . 

after individually labeling of protein samples with iodotmtsixplex 
 reagents as shown in figure 1b , samples are 
 combined before trypsin digestion ( figure 2a ) . since
the labeling of cys thiols by iodotmt reagents is irreversible , 
 reduction with dtt and alkylation with iodoacetamide are possible 
 for more efficient digestion than that of the reversible thiol reactions . 

digested peptides are then subjected to enrichment with anti - tmt antibody 
 resin following the protocol described in materials 
 and methods . because of the low abundance of sno - proteins and 
 sno - cys modifications , it is essential to enrich the iodotmt - labeled 
 peptides before ms analysis . during
lc  ms / ms analysis of enriched 
 peptides , sno - cys are identified by the site of iodotmt labeling and 
 quantified based on ratios of the iodotmtsixplex reporter ions .
after switch labeling with iodotmtsixplex reagents 
 as shown in figure 1b , the six samples are 
 combined for in - solution trypsin digestion and peptide enrichment 
 with anti - tmt resin .
ms / ms 
 for sno - cys site identification and quantification based on iodotmt 
 reporter ions .
( a ) surface plasmon resonance binding curve of anti - tmt binding 
 to tmt - derivatized biacore chip with ( gray line ) and without ( black 
 line ) addition of a competitive tmt analogue compound .
( b ) graph of competitive tmt analogue compounds screened using surface 
 plasmon resonance .
binding data is normalized to mock injections and 
 tmt antibody off rate steady state kinetics at 10 mm concentration 
 of the testing compounds in 500 mm teab buffer , ph 8.5 .
dmac , dimethylacetamide ; 
 dmpz , dimethylpiperizine , tmp , tetramethylpiperidine , mp , methylpiperidine , 
 dmpp , 2,6-dmpp , dipea , diisopropylethylamine .
( c ) comparison of acidic 
 elution buffer eb1 and competitive elution buffer eb2 for enrichment 
 of iodotmt - labeled sno - peptides .
bv-2 cell lysates treated or untreated 
 with snoc ( 200 m ) were processed following the isa workflow . 

ms / ms analysis shows that iodotmt - labeled peptides account 
 for 0.15% , 0.22% , 6.13% , and 21.36% of total peptides in the unenriched , 
 flow through , enriched by eb1 elution , and enriched by eb2 elution , 
 respectively .
a unique feature of the 
 isa workflow is the use of an immobilized 
 anti - tmt resin for labeled peptide enrichment .
since an iodotmt reagent 
 is used to label peptides in the switch assay instead of a biotinylation 
 reagent , endogenously biotinylated proteins are not pulled down during 
 enrichment , resulting in improved specificity of detecting sno - proteins . 

previously , an acidic elution buffer [ 50% ( v / v ) acn/0.4% ( v / v ) tfa ; 
 eb1 ] was used to elute tmt - labeled peptides from the anti - tmt resin 
 after enrichment . however ,
this buffer can also 
 elute a certain amount of nonspecific binding peptides along with 
 the specific tmt - labeled peptides . in order to further improve the 
 specificity of the peptide enrichment while retaining the efficiency 
 of the anti - tmt affinity precipitation , a new elution buffer containing 
 a tmt structural analogue that could specifically compete for tmt - labeled 
 peptide binding was developed .
we screened a series of compounds representing 
 various structural motifs of the tmt tag for testing anti - tmt binding 
 affinity using a biacore competition assay ( figure 2b , a ) .
we found that structural analogues of the mass tag 
 reporter region had the greatest ability to compete for anti - tmt antibody 
 binding ( figure 2b , b ) . comparing the original 
 acidic elution buffer eb1 with the newly developed competitive elution 

buffer ( 10 mm 2,6-dmpp/500 mm teab , ph 8.5 ; eb2 ) for enrichment of 
 the iodotmt - labeled sno - peptides revealed a significant increase in 
 labeled peptide percentage of total identified peptides from 6.13% 
 to 21.36% ( figure 2c ) .
these data show that 
 eb2 works more effectively than that of eb1 and was therefore used 
 in isa for subsequent studies . to demonstrate the efficiency of the isa 
 method , triplicates of untreated and snoc ( 200 m)-treated bv-2 
 microglial cell lysates were analyzed by isa .
figure 3a shows a representative ms / ms spectrum of an identified sno - peptide 
 ( left ; peptide sequence : yvdiaipcnnk ; protein reference : 40s ribosomal 
 protein sa ; sno - cys modification site : c8 ) .
the quantification of 
 this peptide is shown as well ( figure 3a , right ) . 
 in total , 134 sno - cys sites were identified , and they were detected 
 in all of the three replicates .
ninety of them from 68 proteins showed 
 significant changes in s - nitrosylation level ( fold 
 change > 1.3 , p < 0.05 ) when comparing between 
 untreated and snoc - treated samples ( supplemental 
 table s1 , supporting information ) .
for the sno - peptides significantly 
 altered by snoc , we were able to distinguish the modification site 
 on eight peptides containing two cys residues ( figure 3b and highlighted in green in supplemental 
 table s1 ) ; and we also found five peptides with two or more 
 sno - cys modifications per peptide ( figure 3b and highlighted in red in supplemental table 
 s1 ) .
four of the identified proteins were labeled by conventional 
 method bst to validate these lc 
overall , these results show the capability of 
 isa for sno - cys site localization and quantification in multigroup 
 samples .
( a ) bv-2 cell lysates treated or untreated 
 with snoc ( 200 m ) were analyzed by the isa method ( n = 5 ) . the ms spectrum ( left ) and
the coefficients of variation 
 ( cv ) are 10% and 27% for the three replicates of untreated and snoc - treated 
 samples , respectively .
( b ) in total , we identified 90 sno - cys sites 
 on 85 unique peptides exhibiting significant s - nitrosylation 
 level changes ( gray ) and 44 sites on 43 peptides showing low response 
 to snoc treatment ( white ) . among the 90
significantly altered sno - cys 
 sites , 8 multiple cys - containing peptides ( multi - cys peptides ) have 
 only 1 site specifically s - nitrosylated ; 5 multi - cys 
 peptides have 2 sites modified by s - nitrosylation ; 
 and the remaining sno - cys sites were identified in 72 peptides containing 
 a single cys ( 1-cys peptides ) .
( c ) four sno - proteins identified by 
 isa were selected for validation with the bst method .
proteins in 
 bv-2 cell lysates were in vitro s - nitrosylated by 
 snoc exposure ( 0 , 20 , or 200 m ) and then switch - labeled by 
 biotin - hpdp .
after pull - down with avidin , immunoblotting with individual 
 antibodies confirmed these proteins were s - nitrosylated 
 in a dose - dependent manner ( left ) .
total proteins collected before 
 pull - down are shown as loading controls ( right ) . to assess the ability of isa to investigate 
 protein s - nitrosylation in vivo
, 
 we used the method to detect s - nitrosylation in a 
 neuroinflammatory model using bv-2 microglial cells stimulated with 
 lps .
microglia are innate immune active cells in the brain and are 
 associated with inflammatory responses to brain injury and neurological 
 diseases such as alzheimer s disease , parkinson s disease , 
 and stroke .
these cells have been shown to respond to the endotoxin lps to induce 
 protein s - nitrosylation .
after bv-2 cells were exposed to lps ( 100 ng / ml ) 
 for 16 h , the no concentration in the cell culture medium significantly 
 increased ( figure 4a , left ) , and no significant 
 cell death was observed by the mtt assay ( figure 4a , right ) .
biological triplicate lps - treated and untreated 
 bv-2 cells were then lysed in hents buffer , and equal amounts of the 
 lysates were labeled by iodotmtsixplex reagents ( 126 , 128 , and 130 
 for lps - treated samples ; 127 , 129 , and 131 for untreated samples ) . 

after anti - tmt enrichment , the percentage of iodotmt - labeled peptides 
 increased from 0.1% to 35.1% of the total peptides ( data not shown ) .
analysis of protein s - nitrosylation 
 in lps - stimulated 
 bv-2 microglial cells by isa .
( a ) bv-2 cells were exposed to 100 ng / ml 
 lps for 16 h. no production significantly increased after lps - exposure 
 as indicated by griess assay ( left ) . * , p < 0.05 
 vs untreated .
compared to the untreated sample , lps treatment caused 
 2.4% cell death , which is not a significant change ( mtt assay ; right ) . 

( b ) untreated and lps - treated samples were analyzed by isa ( n = 3 ) .
a total of 101 unique sno - cys sites were identified , 
 of which 38 on 36 unique peptides showed significant differences ( fold 
 change > 1.3 , p < 0.05 ) in s - nitrosylation 
 level between untreated and lps - treated samples . to globally visualize 
 the data set , a scatter plot was used to depict each of the sno - cys 
 sites as a point , plotting the log2 fold change against log10 p - value .
the horizontal dashed line demarcates p = 0.05 , and the vertical dashed line demarcates a 1.3-fold change . 

( c ) the quantification information of three representative sno - peptides 
 is presented : ivsnascttnclaplak from protein gapdh [ cv 
 ( untreated ) = 9% , cv ( lps ) = 10% ] , mivecvmnnatctr from 
 protein fabp5 [ cv ( untreated ) = 7% , cv ( lps ) = 5% ] , and vnqigsvteslqack 
 from protein enoa [ cv ( untreated ) = 11% , cv ( lps ) = 5% ] .
a total of 101 unique sno - cys sites were identified , 
 and the site 
 overlap among the three replicates is shown as supplemental figure s2 , supporting information .
to globally 
 visualize the data set , log2 fold change of each sno - cys was plotted 
 against log10 p - value ( figure 4b ) .
sixty - three sno - cys on 60 peptides showed low response 
 to lps stimulation , while s - nitrosylation levels 
 of 38 sno - cys sites on 36 peptides corresponding to 30 proteins were 
 significantly up - regulated ( fold change > 1.3 , p < 
 0.05 ; supplemental table s2 , supporting information ) . among them , two peptides were detected with two sno - cys sites 
 ( highlighted in red in supplemental table s2 ) , and four peptides were identified with one sno - cys and another 
 cys residue with a different modification ( highlighted in green in supplemental table s2 ) .
figure 4c shows the relative quantification 
 of three example peptides that responded to lps treatment .
peptide 
 ivsnascttnclaplak from protein gapdh contains one cys 
 residue at c7 with an iodotmt modification and another cys residue 
 at c11 with carbamidomethyl modification introduced during peptide 
 alkylation by iodoacetamide before trypsin digestion ( figure 4c , top ) .
peptide mivecvmnnatctr from 
 protein fabp5 has two cys residues at c5 and c12 , both of which can 
 be s - nitrosylated but not simultaneously ( figure 4c , middle ) .
we present here a peptide where c12 
 was s - nitrosylated and c5 alkylated by carbamidomethyl . 

enoa peptide vnqigsvteslqack contains only one cys , which 
 was s - nitrosylated ( figure 4c , bottom ) .
the ms / ms spectra of these three peptides are provided 
 in supplemental figure s3 .
these results 
 indicate that isa is able to map sno - cys sites even on peptides containing 
 more than one cys residue and illustrates that this method is efficient 
 in quantitative analysis of protein s - nitrosylation 
 under pathophysiological conditions . to demonstrate the application of isa for comprehensive in vivo studies and to examine the multimodal action of 
 botanical compounds as preventive medicine , we further investigated 
 the effect of sac on s - nitrosylation in lps - stimulated 
 bv-2 microglial cells .
as the most abundant organosulfur compound 
 in age , sac has been reported to act as an antioxidant and show protective 
 effects in many experimental paradigms of neurodegenerative diseases 
 including ischemic and traumatic brain injury , alzheimer and parkinson s 
 disease . since activation of microglia 
 has been implicated in neuroinflammation underlying neurodegenerative 
 diseases , we anticipate that studying the effect of sac on protein s - nitrosylation in activated microglial cells would provide 
 insights into the underlying mechanism(s ) of sac for the attenuation 
 of excessive neuroinflammation and prevention of neurodegenerative 
 diseases . in this study
, bv-2 cells were exposed to lps in the 
 presence or absence of sac ( 5 mm ) or l - name ( 0.5 mm ) , a known 
 inhibitor of no synthases .
our data showed that sac significantly 
 inhibited no production induced by lps but had no apparent toxicity 
 to the cells ( figure 5a ) .
this result is in 
 line with a previous report showing that sac inhibited no production 
 in lps / cytokine - stimulated macrophages .
isa analysis with the biological triplicate lps - treated and lps 
 + sac - treated samples identified 115 sno - cys sites on 110 peptides . 
 the modification site overlap of the three replicates is shown as supplemental figure s4 , supporting information .
the identified sno - cys sites were visualized as points in a scatter 
 plot generated by plotting log2 fold change against log10 p - value ( figure 5b ) .
a total of 46 
 sno - cys sites on 46 proteins displayed significant changes in s - nitrosylation levels ( fold change > 1.3 , p < 0.05 ; supplemental table s3 , supporting 
 information ) .
about 70% of the 46 sno - cys sites have been previously 
 identified in other studies ( data not shown ) .
figure 5c shows relative quantification of three representative iodotmt - labeled 
 peptides whose s - nitrosylation level was down - regulated 
 upon sac treatment .
effect of sac on lps - induced s - nitrosylation 
 in 
 bv-2 cells .
( a ) a griess assay ( left ) showed that no production in 
 bv-2 cells induced by lps was significantly inhibited by sac ( 5 mm ) 
 treatment as well as l - name ( 0.5 mm ) , a known no synthase 
 inhibitor . * , p < 0.05 vs untreated ; # , p < 0.05 vs lps . sac or l - name alone
results of mtt assay ( right ) 
 indicated lps treatment caused about 12.5% cell death ; however , the 
 addition of sac or l - name did not significantly affect the 
 cell viability . ( b )
we identified 115 sno - cys sites with 46 of them 
 differentially s - nitrosylated ( fold change > 1.3 , p < 0.05 ) responding to sac treatment in lps - stimulated 
 bv-2 cells ( n = 3 ) .
the identified sno - cys sites 
 are indicated as dots in a graph generated by plotting log2 fold change 
 against log10 p - value .
the horizontal dashed 
 line is drawn at p = 0.05 , and the vertical dashed 
 line is drawn at fold change = 1.3 .
( c ) the quantitative results 
 of three peptides ytcsfcgk [ cv ( lps ) = 6% , cv ( lps + sac ) = 33% ] , 
 savdncqdswr [ cv ( lps ) = 4% , cv ( lps + sac ) = 13% ] , and qactpmfr 
 [ cv ( lps ) = 8% , cv ( lps + sac ) = 15% ] are given as examples .
in this study , we 
 identified 68 , 30 , and 46 sno - proteins that were differentially s - nitrosylated ( fold change > 1.3 , p < 
 0.05 ) responding to snoc , lps , and lps + sac treatments in bv-2 cells , 
 respectively ( figure 6a ) .
there were 14 sno - proteins 
 shared by snoc- and lps - treated samples , suggesting common and distinct 
 signaling pathways were triggered by these two treatments .
twelve 
 common sno - proteins were found between lps and lps + sac treatments 
 ( figure 6a ) , indicating sac partially attenuated 
 the effect of lps on protein s - nitrosylation but 
 also had its own distinct targets .
nineteen out of 46 sno - proteins 
 responding to lps + sac - treatment were in common with the snoc - treatment . 

the six sno - proteins shared by all of the three groups include lcp1 , 
 pkm2 , slc25a5 , cfl1 , enoa , and ldha .
( a ) 
 the overlaps between the 
 three subsets of sno - proteins identified from different treatment 
 conditions in this study .
( b ) subcellular location of the 46 sno - proteins 
 responding to sac treatment in lps - stimulated bv-2 cells .
( c ) top 
 10 ipa canonical pathways targeted by sac in lps - stimulated bv-2 cells 
 are presented : ( a ) , glycolysis i ; ( b ) , gluconeogenesis i ; ( c ) , eif2 
 signaling ; ( d ) , sucrose degradation ; ( e ) , 14 - 3 - 3-mediated signaling ; 
 ( f ) , tca cycle ii ( eukaryotic ) ; ( g ) , regulation of eif4 and p90s6k ; 
 ( h ) , mtor signaling ; ( i ) , remodeling of epithelial adherent junctions ; 
 ( j ) , glycine biosynthesis i. these pathways were ranked according 
 to their log ( p - value ) ( black bars ) .
a ratio 
 ( gray square ) indicates the number of identified sno - proteins found 
 in each pathway over the total number of proteins in that pathway . 

( d ) the top protein network associated with sac - treatment in lps - stimulated 
 bv-2 cells functions in neurological disease as predicted by ipa . 

twenty - seven identified sno - proteins are involved in this network 
 and displayed in gray .
the color intensity indicates the degree of 
 down - regulation of protein s - nitrosylation .
solid 
 lines in the network imply direct interactions between proteins , and 
 dashed lines indicate indirect interactions .
geometric shapes represent 
 various general functional protein groups ( diamond for enzyme , oval 
 for transcription regulator , trapezoid for transporter , inverted triangle 
 for kinase , double circle for complex / group , and circle for others ) . 

proteins in white shapes are not part of our data set but have relationships 
 with our proteins in the network . in order to learn more about the molecular mechanisms of 

no signaling 
 under microglial activation and of sac to modulate lps - induced s - nitrosylation in bv-2 microglial cells , we conducted functional 
 annotation and pathway analysis of the sno - proteins identified .
ipa 
 analysis shows the 68 sno - proteins significantly altered by snoc treatment 
 are mainly associated with neurological disease , immunological disease , 
 and inflammatory disease , while the 30 sno - proteins in response to 
 lps - stimulation are mostly involved in immunological disease and inflammatory 
 disease / response ( data not shown ) .
other functional annotation results 
 for snoc and lps treatments , including molecular and cellular functions 
 ( supplemental tables s4 and s5 ) , subcellular 
 locations , and top canonical pathways ( supplemental 
 figures s5 and s6 ) are shown in supporting 
 information .
specific effects of sac were assessed using 
 an in - house multicom - pdcn 
 analysis .
the results of this analysis 
 shows that the 46 sno - proteins modulated by sac are mainly located 
 in the cytoplasm and the nucleus ( figure 6b ) , 
 and they are involved in diverse molecular and cellular functions 
 including carbohydrate metabolism , cellular assembly and organization , 
 cellular function and maintenance , molecular transport , and rna trafficking 
 as predicted by ipa ( table 1 ) .
top canonical 
 pathways in which these proteins participate include glycolysis i , 
 gluconeogenesis i , eif2 signaling , sucrose degradation , 14 - 3 - 3-mediated 
 signaling , tca cycle ii ( eukaryotic ) , regulation of eif4 and p90s6k , 
 mtor signaling , remodeling of epithelial adherent junctions , and glycine 
 biosynthesis i ( figure 6c ) . moreover , sac mediated 
 a protein  protein interaction network that functions in neurological 
 disease ( figure 6d ) .
twenty - seven out of the 
 identified 46 sno - proteins are involved in this network .
these findings 
 illustrate that sac exhibits multimodal action on protein s - nitrosylation under microglial activation , which may further 
 contribute to the reversing effects in neurological diseases .
s - nitrosylation is regarded 
 as a major mechanism 
 for no bioactivity with participation 
 in cellular processes , including vasodilation , neurotransmission , 
 apoptosis , cellular trafficking , and cell cycle regulation . to facilitate efficient investigation of s - nitrosylation 
 under physiological and pathophysiological conditions ,
we have developed 
 a novel ms - based isa method based on the conventional bst assay for 
 the detection , identification , and multiplex quantification of sno - cys 
 residues .
we demonstrated in both in vitro and in vivo studies under physiological conditions that isa 
 is an effective tool for investigation of protein s - nitrosylation with several unique features : ( i ) irreversible labeling 
 of sno - cys residues for specific ms detection and site - mapping ; ( ii ) 
 anti - tmt enrichment and competitive elution to improve efficiency 
 of identification of sno - peptides ; and ( iii ) multiplex iodotmt reagents 
 for unbiased quantification of sno - proteins from complex biological 
 samples under different conditions .
there is substantial evidence 
 indicating that s - nitrosylation is a highly conserved 
 process and is precisely regulated 
 in cells . despite the possibility of multiple cys residues 
 in a protein , only specific sites can be s - nitrosylated 
 in response to no signaling and subsequently regulate protein activities . unlike other ptms in proteins ( e.g. , phosphorylation , glycosylation , 
 and sumoylation ) , the linear consensus motif and structural environment 
 that promote protein s - nitrosylation are currently 
 under intensive discussion .
increasing evidence shows a consensus 
 acid  base motif containing an acidic or basic residue flanking 
 the sno - cys sites in the primary amino acid sequence or tertiary structure 
 plays a critical role in protein s - nitrosylation .
proteome - wide studies also show that hydrophobicity of flanking 
 residues , sulfur atom exposure , and cys relative surface accessibility 
 are potential factors contributing to the specificity of s - nitrosylation .
since there are multiple molecular mechanisms for protein s - nitrosylation , it seems that the environments surrounding 
 modified cys residues possess diverse features rather than a general 
 rule that applies to all sno - cys sites . in our study , 
 we identified a total of 133 sno - cys sites from 105 proteins in bv-2 
 cells responding to different stimulations ( snoc , lps , and lps + sac ; supplemental table s6 , supporting information ) .
motif analysis by the motif - x algorithm using the 133 sno - cys sites identified here demonstrated six consensus 
 sequences ( supplemental figure s7 , supporting 
 information ) . in motifs
# 2 , # 3 , and # 6 , modified cys residues 
 are flanked by an acid or basic amino acid , which is in line with 
 the previously found acid 
motifs # 4 and # 5 are 
 consistent with the previously predicted motifs containing hydrophobic 
 amino acids .
the most statistically significant consensus sequence , 
 mxxc in motif # 1 , has not been observed previously , suggesting that 
 the proximal polar amino acids may also play a role in selective cys s - nitrosylation .
given the complexity , prediction 
 of s - nitrosylation 
 sites by computational approaches remains challenging .
therefore , 
 determining the locations of sno - cys sites still relies on proteomic 
 analyses to provide large informative data sets . in previously established 
 snosid and sno - rac methods ,
the labeling of sno - proteins with biotin - hpdp 
 or thiol - reactive resin is a reversible process , and therefore the 
 tags can be lost during peptide elution from the resin at the affinity 
 enrichment step . without labeling , it is difficult 
 to locate the sno - cys sites on peptides with multiple cys residues 
 by ms analysis .
in addition , it is difficult to exclude nonspecific 
 peptides , thus introducing false positive signals .
the peptides linked 
 to sno - peptides by intermolecular disulfide bonds could be one of 
 the  nonspecific  sources , but a reduction step to break 
 the disulfide bonds in protein samples before affinity enrichment 
 is not feasible in these methods due to their reversible nature of 
 sno - cys labeling .
in contrast , the iodoacetyl 
 reaction of iodotmt reagent with s - nitrosothiol used 
 in the isa method is irreversible resulting in a stable tagging modification 
 even under reducing environments .
the iodotmt tag on sno - cys easily 
 removes the ambiguity of site assignment and excludes potential false 
 positive signals from nonspecific peptides in ms analysis .
for example , 
 the gapdh peptide ivsnascttnclaplak , corresponding to 
 residues 144160 , was previously identified by the snosid method as endogenous sno - cys - containing peptide , although 
 the position of the cys residue undergoing s - nitrosylation 
 was unknown . in this study using the isa 
 approach , we successfully determined cys-150 as the s - nitrosylation site but not cys-154 in response to lps treatment 
 ( supplemental table s2 and figure 4c , supporting 
 information ) .
it has been reported that s - nitrosylation 
 of gapdh at cys-150 leads to its binding to siah1 ( an e3 ubiquitin 
 ligase ) which further initiates apoptotic cell death . on the basis of this example , unambiguous sno - cys site - mapping 
 by the isa approach can provide clues and directions for future functional 
 analysis of sno - proteins .
sensitivity is an important criterion 
 when investigating s - nitrosylation due to the labile 
 nature and low abundance 
 of this modification .
however , most existing methods were assessed 
 under very different conditions ( e.g. , concentrations of no donors , 
 cell or tissue types , or treatment conditions ) , resulting in difficulty 
 in comparing sensitivity .
particularly , high doses of no donors , beyond 
 the range of physiological no concentrations , were used in previous 
 proteomics investigations .
for instance , s - alkylating 
 labeling strategy determined 586 sno - cys sites of 384 sno - proteins 
 from 1 mm s - nitroso - n - acetylpenicillamine / l - cysteine - treated mouse ms-1 endothelial cells , and sno - rac identified 396 sno - peptides using 500 m 
 snoc treatment in hek293 cells . even though 
 a large number of sno - proteins were identified , the biological relevance 
 of these data and the sensitivities of these methods under in vivo conditions remain uncertain .
methods using physiological 
 or pathological conditions to induce s - nitrosylation 
 have previously identified up to 90 sno - cys sites . in particular , a recent report using a sequential 
 iodotmt switch strategy to monitor multiple cys modifications identified 
 13 sno - cys on 12 proteins exhibiting significant level changes in s - nitrosoglutathione - treated heart myoblast h9c2 cells under 
 hypoxia conditions . with similar experimental 
 conditions
, we identified 90 , 38 , and 46 sno - cys sites significantly 
 altered by snoc , lps , and lps + sac treatments in bv-2 microglial 
 cells , respectively . the good sensitivity of isa results from its 
 robust workflow with irreversible iodotmt labeling for investigation 
 of protein s - nitrosylation only and the use of competitive 
 elution for efficient anti - tmt peptide enrichment .
as shown in figure 2c , the new volatile competitive elution buffer containing 
 a tmt analogue ( 2,6-dmpp ) significantly increased the percentage of 
 the iodotmt - labeled peptides in total identified peptides ( 21.36% ) 
 compared to that eluted by the nonselective acidic elution buffer 
 ( 6.13% ) , which was used in previous studies .
ms / ms is a powerful method 
 for simultaneous analysis of samples from biological replicates , time 
 courses , or different experimental conditions such as healthy versus 
 disease or various drug treatments .
previously , ms quantification 
 of s - nitrosylation has been accomplished using isotope - coded 
 affinity tags ( icat ) labeling , s - nitrosothiol 
 capture ( snocap ) reagents , isobaric tag 
 for relative and absolute quantitation ( itraq)-coupled sno - capture 
 techniques , or cystmt switch labeling .
icat and snocap are stable isotope labeling methods 
 for comparing relative peptide abundance at the ms level but are limited 
 to the analysis of two samples at a time .
moreover , the quantification 
 of peptide ratios using ms peak intensities may be unreliable due 
 to the shift of elution time between the light and heavy labeled counterparts 
 on the rp - lc separation by icat and snocap .
in contrast , isobaric tags with the identical mass and chemical 
 structures ( e.g. , itraq and tmt ) allow for multiplexing up to 8 or 
 10 samples in a single lc  ms / ms analysis .
amine - reactive itraq 
 tags have been used for labeling sno - peptides , but as there is no 
 mass signature on sno - cys , the sno - rac workflow is unable to localize 
 sno - cys sites despite ms quantification of the enriched peptides . utilizing isobaric cystmtsixplex reagents in 
 the cystmt
however , the output of this method is relatively 
 low for cell - based study , as only 25 sno - cys sites were detected from 
 snoc ( 200 m)-treated cells .
since cystmt labeling of sno - proteins 
 is reversible , it is not compatible with reduction / alkylation , which 
 is required for complete protein denaturation before trypsin digestion . 

this may result in incomplete digestion in complicated protein samples 
 therefore hindering the efficiency of ms detection .
unlike cystmt , 
 the iodotmt reagents irreversibly label cys thiols for improved labeling 
 of sno - proteins .
coupled with the improved competitive elution buffer 
 for anti - tmt enrichment , the isa workflow allows for multiplex s - nitrosylation sample analysis with high efficiency and 
 specificity .
emerging evidence indicates that protein s - nitrosylation 
 exerts critical roles in a variety of cellular processes by regulating 
 protein folding , ubiquitination , mitochondrial dynamics , and signal 
 transduction .
many neurodegenerative diseases 
 involve nitrosative stress and proinflammatory responses including 
 protein s - nitrosylation with activation of microglial 
 cells .
our investigation 
 of the s - nitrosylation proteome using isa in lps - stimulated 
 bv-2 cells provided insights into the no signaling pathways under 
 microglial activation and shed light on possible molecular mechanisms 
 contributing to neurological diseases .
ipa results showed that several 
 mitochondrial metabolic pathways ( e.g. , glycolysis i , gluconeogenesis 
 i , and tca cycle ii ) are altered by s - nitrosylation 
 in lps - stimulated bv-2 microglial cells ( supplemental 
 figure s6 , supporting information ) .
these results are consistent 
 with a recent report in which a number of enzymes that regulate these 
 metabolic pathways were found to be s - nitrosylated 
 in different mouse organs .
there are 
 also other studies showing that glycolysis was increased in lps - induced 
 bv-2 cells , and monocyte - derived inflammatory 
 dendritic cells committed to glycolysis to maintain atp levels when oxidative phosphorylation 
 was inhibited by no .
it is possible that s - nitrosylation of the enzymes in glycolysis and other mitochondrial 
 metabolic pathways identified here are involved in the regulation 
 of these pathways during inflammatory response .
administration of sac to 
 cells before lps induction of s - nitrosylation was 
 able to restore the effect of lps on glycolysis i and gluconeogenesis 
 i ( figure 6c ) , suggesting a protective effect 
 for sac on mitochondrial metabolism .
to our knowledge , this is the 
 first time sac has been linked to glycolytic / gluconeogenic metabolism 
 regulation .
our data also showed that sac altered protein s - nitrosylation in the eif2 ( eukaryotic initiation factor 
 2 ) signaling pathway , which is known to be an important pathway controlling 
 translation initiation of cellular recovery genes in response to various 
 stresses in eukaryotic cells and is associated with pathogenesis of 
 neurodegenerative diseases including alzheimer s disease .
sac may act through eif2 signaling as well as other neurological 
 disease - related pathways identified here ( figure 6c ) , such as mtor- and 14 - 3 - 3-mediated signaling , to take part in nitrosative stress defense .
collectively , 
 our data demonstrate that the isa method is a powerful 
 tool to profile protein s - nitrosylation under physiological / pathological 
 conditions .
isa provides a simple solution to the multiple problems 
 existing in previous methods for s - nitrosylation 
 analysis . with high specificity , high sensitivity , and site - mapping / quantification 
 ability ,
isa well meets many of the current demands for the investigation 
 of protein s - nitrosylation .
this approach could have 
 broad applications in the determination and comparison of s - nitrosylation proteome under various conditions .
it can 
 be used to help further our understanding of molecular mechanisms 
 for no signaling and progression of diseases .
moreover , it may also 
 facilitate identifying potential therapeutic targets for disease treatment 
 and elucidating effects of drugs .
 s - nitrosylation is a redox - based protein post - translational 
 modification in response to nitric oxide signaling and is involved 
 in a wide range of biological processes . 
 detection and quantification 
 of protein s - nitrosylation have been challenging 
 tasks due to instability and low abundance of the modification . 
 many 
 studies have used mass spectrometry ( ms)-based methods with different 
 thiol - reactive reagents to label and identify proteins with s - nitrosylated cysteine ( sno - cys ) . in this study , we developed 
 a novel iodotmt switch assay ( isa ) using an isobaric set of thiol - reactive 
 iodotmtsixplex reagents to specifically detect and quantify protein s - nitrosylation . 
 irreversible labeling of sno - cys with the 
 iodotmtsixplex reagents enables immune - affinity detection of s - nitrosylated proteins , enrichment of iodotmt - labeled peptides 
 by anti - tmt resin , and importantly , unambiguous modification site - mapping 
 and multiplex quantification by liquid chromatography  
 tandem 
 ms . 
 additionally , we significantly improved anti - tmt peptide enrichment 
 efficiency by competitive elution . using isa 
 , we identified a set 
 of sno - cys sites responding to lipopolysaccharide ( lps ) stimulation 
 in murine bv-2 microglial cells and revealed effects of s - allyl cysteine from garlic on lps - induced protein s - nitrosylation in antioxidative signaling and mitochondrial metabolic 
 pathways . 
 isa proved to be an effective proteomic approach for quantitative 
 analysis of s - nitrosylation in complex samples and 
 will facilitate the elucidation of molecular mechanisms of nitrosative 
 stress in disease . 
Introduction
Materials
and Methods
Results
Discussion