chromatin is a complex of dna and proteins , in which the histones h2a , h2b , h3 , and h4 are the major protein constituents [ 1 , 2 ] . chromatin remodeling through posttranslational modification of histones plays an important role in modulation of dna - protein interaction and thus regulates various biological processes such as replication , dna damage repair , and transcription . hence , identification of the chromatin associated nonhistone proteins ( chraps ) would permit understanding the molecular mechanisms for chromatin remodeling and regulation of various biological processes . fission yeast is a useful model for analysis of rna interference ( rnai ) directed heterochromatin formation [ 4 , 5 ] . many chraps have been identified by using the high - throughput proteomic analysis of protein complexes purified through the chromatin immunoprecipitation ( chip ) coupled with the tandem affinity protein purification ( tap ) tagging method in which the known chrap is used as bait [ 612 ] . however , it is limited to the identification of the chraps that are associated with the complexes containing the previously known chraps . a traditional assay for testing whether a protein of interest is associated with the chromatin includes the preparation of chraps extracts ( or chrap - prep ) through collection of the released proteins from the dnasei digested crude chromatin and western blot analysis [ 1316 ] . by using this method , components of the origin recognition complex such as orc1 , orc2 , and orc5 are found to be associated with the chromatin throughout the cell cycle [ 13 , 14 ] . on the other hand , the atr - like kinase rad3 and the mitotic activator phosphatase cdc25 are found to be temporally associated with chromatins upon dna damage [ 15 , 16 ] . the relative level of a chrap of interest in chrap - prep is clearly higher than that of whole cell extract ( wce ) [ 1316 ] . nevertheless , it is unclear if highly abundant cytoplasm - localized proteins are effectively depleted from the chrap - prep . effectively , depletion of the highly abundant non - chraps such as ribosomal proteins is essential for de novo identification of chraps through proteomic analysis of chrap - prep based on their absolute abundances . we found that the top ranked proteins by levels of abundance in chrap - prep were predominated by the ribosomal proteins , suggesting that the highly abundant non - chraps are not effectively removed in chrap - prep . hence , simply based on the level of protein abundance in chrap - prep by using the high - throughput proteomic analysis is unlikely to reveal chrap candidates without prior knowledge . silac ( stable isotope labeling with amino acids in cell culture)-mediated proteomic analysis has shown to permit the quantitative analysis of the relative protein levels between those labeled with and without heavy isotopes , allowing estimation of ratios between individual protein levels in chrap - prep versus wce . by applying the silac - mediated high - throughput proteomic analysis , we show , in this study , that the highly abundant non - chrap ribosomal proteins are significantly depleted in the top ranked proteins by silac - ratio between chrap - prep and wce . the top ranked proteins by silac - ratio are enriched for potential chrap candidates such as nucleus - localized proteins and chrap - associated functions such as chromatin structure organization , dna replication and repair , transcription according to gene ontology analysis . phenotypic assessment shows that the silac - enriched but not depleted nucleus - localized proteins exhibit the high likelihood of requirement for growth fitness in mms ( methyl methanesulfonate)-induced dna damage stress . taken together , our results indicate that silac - mediated proteomic analysis of chrap - prep is capable of identifying chrap candidates without prior knowledge . we propose that our approach can be complementary to the chip method coupled with tap - tagging for identification of chrap - interacting partners . in the silac proteomic analysis , silac - labeled samples are required to be fully incorporated with the heavy stable isotope - coupled lysine ( e.g. , c6-lysine or heavy - lysine ) or arginine ( e.g. , c6-arginine or heavy - arginine ) or both . to avoid the arginine - conversion problem the rate of incorporation with heavy - lysine in cells after various numbers of passages or subcultures in minimal medium supplemented with heavy - lysine was tested . in this test , each subculture was maintained for a day ( i.e. , equivalent to ~3 generations ) before subsequent subculturing ( figure 1(a ) ) . the relative level of light versus heavy peptides / proteins was exemplified by the eno1 peptide avgnvnniiapvvk in various subcultures . as expected , no eno1 peptide was detected to contain heavy - lysine in the initial culture ( p0 ) prior to subculturing with the heavy lysine - containing medium ( figure 1(b ) , see p0 ) . on the other hand , ~90% of the peptides were detected to contain heavy - lysine in the first subculture ( p1 ) with the heavy lysine - containing medium ( figure 1(b ) , see p1 ) . hardly any light lysine - containing peptides were detected in the second ( p2 ) or the third ( p3 ) subcultures ( figure 1(b ) , see p2 and p3 ) . this result indicates that heavy lysine is effectively incorporated into cellular protein in fission yeast . to further test whether the heavy lysine was uniformly incorporated in all peptides / proteins besides eno1 , a slice of sds - page gel containing proteins derived from the first subculture was subjected to lc - ms / ms analysis . the ratio between light and heavy peptide levels in all of the ~100 peptides detected was found to be close to 3 in log  2 scale , indicating that the heavy lysine is uniformly incorporated in all proteins in the culture ( figure 1(c ) ) . to ascertain proteins were fully incorporated with heavy lysine , cells derived from the third subculture ( i.e. , ~9 generations in the heavy - lysine - containing medium ) were applied for silac analysis in this study . next , we wanted to assess the sensitivity by varying the ratios of light and heavy peptides / proteins in the given premixed samples . a slice of sds - page gel ( i.e. , containing > 100 peptides ) from each given sample was subjected to the proteomic analysis after in - gel trypsinization . to this end , the distribution of ratios of all peptides detected in silac analysis correlated well with the expected ratio in the given samples , suggesting that our silac protocol is adequate for the quantitative proteomic analysis ( figure 2(a ) ) . this is consistent with the notion that silac methodology is excellent for quantitative proteomic analysis . the correlation was apparent when the medians of ratios of all peptides detected in silac analysis were compared to the expected ratios of the given samples ( figure 2(b ) ) . to estimate the ratios between light and heavy proteins , based on this estimation , protein ratios detected in silac analysis correlated well with the expected ratios of the given samples ( figure 2(c ) ) . hence , the median of unique peptide levels was used to estimate the level of proteins in this study . it has been shown that a given proteins can be tested for its association with the chromatins by enriching the chraps [ 13 , 14 ] . based on the protocols , we obtained chrap - prep ( figure 3(a ) ) . it was clear that soluble proteins such as tubulin were depleted only in the soluble fraction ( sup1 ) and chromatin - associated proteins such as histone h4 were enriched in the chrap - prep ( or sup2 ) ( figures 3(b ) and 3(c ) ) . to test if the most abundant proteins in chrap - prep were chraps , chrap - prep was subjected to the non - silac protein analysis . we found that the top 10% ranked proteins by absolute abundance in chrap - prep were overrepresented by the non - chrap ribosomal proteins when compared to the background level ( i.e. , 51.4% versus 14.9% ; p value = 2.21e  07 ) ( see supplementary table s3 in supplementary material available online at doi:10.1155/2012/630409 and also see section 4 ) . this result indicates that the highly abundant non - chraps ribosomal proteins are not effectively removed in chrap - prep . we judged that the absolute abundance of the majority enriched chraps might not be higher than that of the depleted ribosomal proteins due to their high abundance prior to the enrichment . on the other hand , chraps would be top ranked by comparing levels of enrichment or ratios between chrap - prep ( after enrichment ) and wce ( before enrichment ) . to test this possibility , equal amounts of chrap - prep and heavy lysine - labeled wce proteins were mixed and subjected to lc - ms / ms analysis using ltq orbitrap mass spectrometry ( see section 4 ) . a total of 507 proteins were identified based on the presence of paired light ( i.e. , chrap - prep ) and heavy ( i.e. , wce ) peptides after selection of high quality peptide through the trans - proteomic pipeline ( http://tools.proteomecenter.org/ ) ( supplementary table s2 ; see section 4 ) . by ranking the proteins using silac - ratios , we found that the occurrence of ribosomal proteins in the top 10% ( or top 50 ) ranked proteins was ~35% lower than the background level ( 6% versus 9.5% ) . we found that the ribosomal protein occurrence in the top 10% ranked proteins by silac ratios was significantly lower than that in the top 10% ranked proteins by absolute abundances ( 6% versus 51.4% ; p - value = 9.32e  12 ) . this result indicates that the abundant ribosomal proteins can be effectively reduced from the top 10% ranked proteins in silac analysis ( figure 3(d ) ) . we noted that the occurrence of ribosomal proteins in the second top 10% ranked proteins by silac ratios was not reduced when compared to the background level ( i.e. , 14% versus 9.5% ) . this result suggests that the enrichment for chraps in the second top 10% ranked proteins was less effective . hence , only the first top 10% ranked proteins by silac ratio were considered for enriching chrap candidates and were further analyzed in this study . we , therefore , wanted to examine if the nucleus - localized proteins were particularly enriched in the top ranked proteins by silac ratios . gene ontology ( http://www.geneontology.org/ ) , we found that the nucleus - localized proteins were significantly enriched in the top 10% ranked proteins by silac ratios when compared to the background level ( i.e. , 24% versus 9.47% ; p - value = 2.0e  03 ) ( figure 4(a ) ) . this result supports the notion that the top 10% ranked proteins by silac ratios are enriched for charp candidates , suitable for chrap discovery without prior knowledge . next , we wanted to know if the chrap - associated functions such as chromatin modification and dna replication and repair would be enriched in the top ranked proteins by silac ratios . based on the gene ontology ( i.e. , biological process terms ) , gene functions such as transcription , chromatin modification , dna replication and repair were over - represented in the top 10% ranked proteins ( p value < 0.05 ) ( figure 4(b ) ) . these results are consistent with a notion that the top ranked proteins by silac were enriched for chrap functions . in the top 10% ranked proteins by silac ratios , we found psm3 , cbh2 , and c27f1.06c that are involved in chromosome organization and chromatin remodeling ; ddb1 , msh3 , spp1 , and uve1 that are involved in dna replication and repair , and eri1 , c947.08c , rpb1 , and c530.05 that are involved in transcription ( see supplementary table s2 ) . these proteins would represent a small subset of chraps that are relatively soluble , abundant , and constitutively associated with the chromatin . deep analysis of chrap - prep using silac proteomics should allow identifying more proteins with chrap - associated functions without prior knowledge . we also found prp16 , smb1 , and mug161 that are involved in proteolysis and ubr1 , p8b7.11 , and rpt2 that are involved in mrna splicing in the top ranked proteins by silac ratios . in fact , it is not unusual to find components of the proteolysis machinery that are associated with the chromatin . for instance , the ubiquitin e3 ligase pcu4 is found to be associated with the rna - induced transcriptional silencing ( rits ) complex involved in heterochromatin assembly ; and the e3 ligase ubr1 is associated with the set1 complex involved in histone h3 methylation [ 21 , 22 ] . on the other hand , proteins involved in mrna splicing we noted that some nucleus - localized proteins were not listed in the top 10% ranked proteins by silac ratios . those nucleus - localized proteins might not be associated with the chromatins . to test this possibility , randomly selected 3 top silac - ranked proteins msh3 , prp16 , and c18.05c ( e.g. , their silac ratios were 5.86 , 4.72 , and 4.19 in log  2 scale , resp . ) and 2 other proteins srp2 and kap95 ( e.g. , their silac ratios are 0.46 and 1.30 in log  2 scale ) ( see supplementary table s2 ) were subjected to the traditional assay for chraps ( see section 4 ) . the analysis indicated that the silac most enriched nucleus - localized proteins were the true chraps ( figure 5(a ) ) . on the other hand , the silac depleted nucleus - localized proteins were not chraps ( figure 5(b ) ) . to ascertain that the subcellular localization of the ha epitope - tagged protein used in the traditional chrap assay would not be altered by the epitope hence , we conclude that the silac enriched nucleus - localized proteins are chrap candidates . some of the dual - localized proteins were found in the top ranked proteins by silac ratios ( see supplementary table s2 ) . to test if they were the true chraps , the 3 randomly selected proteins uve1 , the analysis indicated that all 3 proteins exhibited apparent enrichment in the chrap - prep when compared to wce ( figure 5(d ) ) . on the other hand , the presence in the soluble fraction was detected in 2 out of 3 dual - localized proteins , consistent with their dual subcellular localization . this result indicates that most of the top ranked proteins by silac ratios are true chrap candidates . of the 507 proteins identified in the silac analysis , 413 were found to be either silac enriched ( i.e. , log  2 silac - ratio > 0.585 ) or depleted ( i.e. , log  2 silac - ratio < 0.585 ) . we wanted to test if the silac - enriched proteins have a likelihood of requirement for growth fitness in dna damage stress , one of the chrap - associated functions . for this reason , 188 ( ~45.5% ) s. pombe gene deletion strains from the bioneer deletion strains set ( version 1 ) were subjected to the phenotypic assessment using the mini - growth curve assay . level of growth fitness under mms stress was estimated by the growth fitness score   ( gfsmms )  that was calculated based on the difference of  t50 ( the time at the half - maximal concentration ) between cultures supplemented with and without mms ( see section 4 ) . the growth fitness score gfsmms was proportional to the level of requirement for growth fitness under mms stress . it was apparent that , among the nucleus - localized proteins , the median gfsmms of the silac - enriched proteins was significantly higher than that of the silac - depleted ones ( 1.43 versus 0.79 ; p value < 0.05 ; supplementary table s4 ) . this result indicates that the top silac - ranked nucleus - localized proteins are bona fide chrap candidates that exhibit a high likelihood of requirement for growth fitness under dna damage stress ( figure 6(a ) ) . on the other hand , among the cytoplasm - localized or dual - localized proteins , the median gfsmms of the silac - enriched proteins showed no apparent differences from that of the silac - depleted ones ( i.e. , 0.93 versus 0.98 or 1.05 versus 1.02 ) ( figures 6(b ) and 6(c ) ) this is consistent with the observation that the cytoplasm - localized or dual - localized proteins were not overrepresented in the top ranked proteins by silac ratios ( see figure 4(a ) ) . hence , silac proteomic analysis of chrap - prep is capable of identifying chrap candidates without prior knowledge . we noted that , however , hardly any chromo - domain or bromo - domain containing chromatin remodelers are found in our silac analysis of chrap - prep . this is probably a result of using the physiological salt concentration in this analysis ( see section 4 ) . it is known that extraction of chromo - domain and bromo - domain proteins requires high salt concentrations . alternatively , these proteins could also escape the detection in lc - ms / ms due to their relatively low abundance and high level of interference from the cytoplasm - localized abundant proteins . it could be improved by better chromatographic separation of the trypsinized peptides using a longer column or uplc separation before mass spectrometric analysis . modification of chrap preparation and improvement of silac proteomic analysis should allow identification of the chromo - domain and bromo - domain proteins in future studies . we show that the chrap - prep used in traditional assays for chraps is predominated by the abundant cytoplasmic proteins such as ribosomal proteins based on the absolute abundance of proteins . on the other hand , we show that proteomic analysis of chrap - prep together with the silac - labeled wce is able to effectively deplete the ribosomal proteins from the top ranked proteins by silac ratios . significantly , we show that the top ranked proteins by silac ratios enrich for nucleus - localized proteins that display a high likelihood of requirement for growth fitness under dna damage stress . hence , the silac - mediated proteomic analysis is capable of determining chraps without prior knowledge . we propose the method shown in this study can be complementary to the proteomic analysis of protein complexes purified via chip with tap - tagged chraps for identification of chrap - interacting partners . strains used in this study are listed in table 1 except for bioneer deletion strains ( bioneer corporation , daejeon , korea ) . the strain lys1 - 131 was used in preparation of the silac - labeled cells . hemagglutinin ( ha)-tagged strains for western blot analysis were constructed based on the protocol reported previously . cultures in minimal medium ( mm ) supplemented with normal or heavy lysine ( c6-lysine ; cat . clm-2247 - 0.25 ; cambridge isotope laboratories , andover , ma , usa ) was used in proteomic analysis . cultures in rich medium ( yes ) supplemented with or without methyl methanesulfonate ( mms ) at the final concentration of 1  mm were used in growth fitness assays . to enrich the chraps , chrap - prep was obtained as described elsewhere with some modification [ 13 , 14 ] . in brief , ~500  ml ( for lc - ms / ms analysis ) or 50100ml ( for western blot analysis ) log - phase growth cells ( od600 = ~0.8 )  were harvested and washed once with stop buffer ( 0.9% nacl , 1  mm nan3 , 50  mm naf , 10  mm edta ) . the washed cells were then protoplasted by resuspending in protoplast buffer ( 35.5  mm bme , 50  mm sodium citrate , 40  mm edta , and 1.2  m sorbitol ) supplemented with 8  mg / ml zymolyase-20 t ( mp biomedicals inc . , protoplasting of cells were monitored frequently under microscope ( protoplasts turned dark when treated with 1% sds ) . the reaction was stopped by addition of an equal volume of ice - cold 1.2  m sorbitol ph 7.5 when ~90% of the cells were protoplasted . the washed protoplasts were resuspended in 450  l 1.2  m sorbitol for generating chraps - prep or snap - frozen in liquid nitrogen and stored at 80c for later use . the resuspended protoplasts were lysed by addition of 50  l 10x lysis buffer ( 500  mm kac , 20  mm mgcl2 , 200  mm hepes ph 7.9 ) supplemented with the 1x complete protease inhibitors edta - free tablet ( roche , basel , switzerland ) and 1% triton x-100 ( tx-100 ) ( promega , fitchburg , wi , usa ) . supernatant ( sup-1 ) containing soluble proteins was transferred to a fresh tube and the pellet ( pel-1 ) was washed twice and resuspended in lysis buffer without tx-100 to yield the crude chromatin extract . the resulting crude chromatin fraction was digested with dnasei ( stratagene , la jolla , ca , usa ) at the concentration of 10 unit/g dna in digestion buffer ( 40  mm tris - hcl ph  7.5 , 6  mm mgcl2 , 150  mm nacl , 2  mm cacl2 and protease inhibitors ) at 37c for 30  min with vigorous shaking.the dnasei - digested crude chromatin extract was centrifuged at 14,000  g for 5  min . the supernatant ( sup-2 ) was referred as the chrap - prep and separated from the pellet ( pel-2 ) . a desired amount of proteins was taken and mixed with standard loading buffer for sds - page analysis . proteins in gel were electrotransferred onto nitrocellulose membranes for probing with primary antibodies against ha ( santa cruz biotechnology , santa cruz , ca , usa ) , histone h4 ( upstate biotechnology , lake placid , ny , usa ) , and -tubulin ( tat1 antibody ; a gift of k. gull , university of oxford , london , uk ) . secondary antibodies conjugated with horse radish peroxidase ( ge healthcare , piscataway , nj , usa ) were used for detection of chemiluminescence signals using the ecl plus system ( ge healthcare ) . gels were sliced into ~50 pieces from top to bottom of a lane . proteins in gel slices were destained and trypsinized in - gel in 25  mm nh4hco3 supplemented with 12.5  ng/l trypsin ( promega , madison , wi , usa ) . the resulting peptides were cleaned using c18 ziptip ( millipore , medford , ma , usa ) and ready for mass spectrometry analysis . mass spectrometry analysis was performed using a nanoflow high - performance liquid chromatography ( hplc ) system ( eksigent , dublin , ca ) connected to a hybrid ltq - orbitrap ( thermo scientific , bremen , germany ) equipped with a nanoelectrospray ion source ( thermo scientific ) . the peptides were separated with a 15  cm long and 75  m inner diameter picofrit column with an integrated tip ( new objective inc , woburn , ma ) packed with 4  m reverse - phase c12 resins ( jupiter proteo phenomenex , torrance , ca , usa ) . hplc mobile phase consists of ( a ) 2% acetonitrile 0.1% formic acid and ( b ) 98% acetonitrile 0.1% formic acid . approximately 10  l peptide solution was loaded onto a nano trap column ( 300sb - c18 , agilent ) with 100% mobile phase a and washed for 10  min at a flow rate of 20  l / min . the trap column was then brought in - line with the nano column using the cn2 nano volume switching valve ( vici valco cheminert , switzerland ) and the peptides were eluted by 235% mobile phase b over 70  min and 3590% over 6  min with a constant flow rate of 300  nl / min . finally the column was washed for 10  min with high concentration of organic solvent ( 90% mobile phase b ) and re - equilibrate with another 15  min with 98% mobile - phase a prior to loading of the next sample . eluted peptides from hplc column were directly electrosprayed into the ltq - orbitrap mass spectrometer for analysis . the spray voltage was set to 2.0  kv and the temperature of the heated capillary was set to 250c . the ms instrument was operated in a data - dependent mode by automatically switching between the full survey scan and ms / ms acquisition . high resolution precursor spectra ( m / z 3002,000 ) were acquired in the orbitrap with resolution of 60,000 at m / z 400 ( after accumulation to a target value of 10 ions in the linear ion trap ) . the 5 most intense ions with ion intensity above 1,000 counts and charged state  2  were sequentially isolated for fragmentation in the linear ion trap using collision induced dissociation ( cid ; normalized collision energy 35% , activation q 0.250 , and activation time 30  ms ) at a target value of 10,000 ions . the dynamic exclusion list was restricted to a maximum retention period of 90  sec and a relative mass window of 10  ppm . the ms and ms / ms spectra were recorded by the mass spectrometer as raw files using the xcalibur software 2.0sr2 ( thermo fisher scientific ) . tpp ( trans - proteomic pipeline version 4.2.1 , seattle proteome centre , institute of systems biology , seattle , wa , usa ) was used to perform database searching and peptide assignment and validation . for this purpose uninterpreted ms / ms spectra were searched against the s. pombe protein databases uniprot knowledgebase , including swiss - prot and trembl using sequest algorithm . the database includes forward protein ( target ) and reverse protein ( decoy ) sequences that were generated by bioworks 3.3.1 ( thermo scientific ) . search parameters used in this study were the requirement of tryptic cleavage ( allowing 1 missed cleavage site ) , minimum peptide length of 7 amino acids , maximal precursor ions mass deviation of 10  ppm , peptide mass tolerance of 0.5  da , static modification on cys of   + 57.0215  da , differential modification on met of 15.9945  da , and heavy isotope coupling on lys of   + 6.0201  da . all assigned peptides were validated using peptideprophet   and the cutoff was set to the probability of 90% or greater . the elution profile of the c6-lys and c6-lys containing peptides from the qualified peptides were isolated and quantified based on the area of peaks by xpress . a total of 507 proteins with a ratio between light ( chrap - prep ) and heavy ( wce ) protein levels were listed in supplementary table s2 . to test whether the chrap candidates ( e.g. , nucleus - localized proteins ) could be dominated in the most abundant proteins ( i.e. , based on the absolute abundance ) in the chrap - prep , proteomic analysis of the chrap - prep proteins without silac was performed . in determination of the absolute abundance , the peptideprophet - qualified peptides ( probability  90% ) were quantified by pepquan and the abundance of proteins was estimated by the median abundance of the respective unique peptides . a total of 376 proteins whose abundance level was approximated by the median level of their unique peptides were listed in supplementary table s3 . based on the absolute abundance , nucleus - localized proteins were not overrepresented in the top 10% most abundant proteins ( supplementary figure s1 ) . notably , on the other hand , the ribosomal proteins were dominated in the top 10% most abundant proteins , suggesting that the highly abundant non - chrap ribosomal proteins are not effectively depleted in the chrap - prep . of 507 proteins identified in the silac - mediated analysis , 413 were either enriched or depleted ( i.e. , ratio  1.5 fold ) in chrap - prep when compared to wce . out of 413 deletion strains , 188 ( 45.5% ) were found in the bioneer deletion strain collection ( version 1 , bioneer corporation ) . therefore , 188 deletion mutant strains ( see supplementary table s1 ) were subjected to minigrowth curve assays using the bioscreen miniculture growth curve system ( growth curves usa , piscataway , nj ) for growth fitness in 1  mm mms stress . growth fitness score in mms ( gfsmms ) is calculated based on the formula ( gfsmms ) = 100(t50_mut/t50_wt ) ,  in which t50 is the time at the half - maximal cell concentration based on the growth curve , and t50 is the difference of the half - maximal time between cultures supplemented with and without mms . binomial test was used to test the nonrandom distribution . in analysis of enrichment or depletion of the nuclear or cytoplasmic proteins , the protein localization information in a genome - wide study by matsuyama et al localization of additional 321 proteins is based on the go terms ( i.e. , cellular components ; http://www.genedb.org/ or http://amigo.geneontology.org/ ) . the subcellular localizations are briefly categorized into three types : the nucleus ( it includes the nucleus , nucleolus , spindle pole body , and nuclear envelope ) , cytoplasm ( it includes cytosol , mitochondrion , golgi , er , cytoskeleton , plasma membrane , and other cytoplasmic organelles ) , or both . thus , 4,708 proteins ( i.e. , ~93% of s. pombe proteome ) have well defined localization types : 763 ( ~16.2% ) are localized at the nucleus , 2180 ( 46.3% ) at the cytoplasm , and 1765 ( 37.5% ) at both the nucleus and cytoplasm . biological process terms were only considered in gene ontology analysis when the occurrence was 15 or greater in the group of 300500 proteins . unpaired two - sample t - test was used to test the difference of gfsmms between the silac - enriched and depleted proteins with nucleus or cytoplasm or both localizations . original mass spectrometric data are deposited in tranche database ( https://proteomecommons.org/ ) and can be accessed by a hash  ap/3mk3fjf/4ay8sslwnzqzdi9fdeoawmmb9oz3th0cnj807urbpmsg / s+hwzm4mazdrmmmhqxi10xe5jo2jaqtfsseaaaaaaaaemq==  or at the author 's website at http://pombe.gis.a - star.edu.sg/.
chromatin - associated nonhistone proteins ( chraps ) are readily collected from the dnasei digested crude chromatin preparation . in this study , we show that the absolute abundance - based label - free quantitative proteomic analysis fail to identify potential chraps from the chrap - prep . 
 this is because that the most - highly abundant cytoplasmic proteins such as ribosomal proteins are not effectively depleted in the chrap - prep . 
 ribosomal proteins remain the top - ranked abundant proteins in the chrap - prep . on the other hand , we show that relative abundance - based silac - mediated quantitative proteomic analysis is capable of discovering the potential chraps in the chrap - prep when compared to the whole - cell - extract . 
 ribosomal proteins are depleted from the top silac ratio - ranked proteins . 
 in contrast , nucleus - localized proteins or potential chraps are enriched in the top silac - ranked proteins . 
 consistent with this , gene - ontology analysis indicates that chrap - associated functions such as transcription , regulation of chromatin structures , and dna replication and repair are significantly overrepresented in the top silac - ranked proteins . 
 some of the novel chraps are confirmed using the traditional method . 
 notably , phenotypic assessment reveals that the top silac - ranked proteins exhibit the high likelihood of requirement for growth fitness under dna damage stress . taken together , 
 our results indicate that the silac - mediated proteomic approach is capable of determining chraps without prior knowledge .