hepatocellular 
 carcinoma ( hcc ) is the sixth most common malignancy 
 and the third most common cause of cancer - related death worldwide . hcc generally develops in patients that already 
 have liver cirrhosis where this group needs to be monitored clinically 
 in order to detect an early onset of hcc . hcc may develop in patients 
 either with alc ( alcohol)- , hepatitis b virus ( hbv)- , or hepatitis 
 c virus ( hcv)-related cirrhosis . effective treatments for hcc 
 include tumor resection , liver transplantation , 
 or percutaneous treatment . in the united states , europe , and japan , these treatments can be applied to only 30% of the patients ( who are 
 diagnosed at early stage ) , and they result in a 5 year survival rate 
 higher than 50% . in comparison , for late stage patients where these 
 treatments can not be applied , the 2 year survival rate is lower than 
 16% . methods for noninvasive detection and monitoring 
 of hcc include 
 imaging ( ultrasound , computer tomography , and magnetic resonance imaging ) 
 and serum marker analysis . however , because 
 the interpretation of imaging is operator - dependent and can be very 
 difficult for persons who are obese , who have underlying cirrhosis , 
 or who have small tumors , reliable serum markers are needed to complement 
 these imaging procedures . serum alpha - fetoprotein ( afp ) is most widely 
 used as a clinical hcc diagnostic marker because of its relatively 
 high auc value ( area under the curve of roc ) of 0.82 between hcc and 
 chronic liver disease samples ; at the cutoff value of 20 ng / ml , it 
 has a specificity of 90.6% and a sensitivity of 60.0% . des - gamma carboxy prothrombin ( dcp ) is widely used as a 
 marker in japan . the diagnostic value of dcp varies among different 
 groups of patients . in patients with small ( <3 cm ) tumors , dcp 
 has an auc value of 0.732 , lower than that of afp ( 0.870 ) ; in patients 
 with large ( > 5 cm ) tumors , it has an auc value of 0.985 , higher 
 than 
 that of afp ( 0.934 ) . in addition , in patients 
 with viral etiology dcp , it has an auc value of 0.76 ; in patients 
 with nonviral etiology , it has an auc value of 0.65 . a recent large clinical study has shown that afp and dcp 
 are complementary but neither is sufficiently accurate to be used 
 for hcc surveillance . hence , new 
 diagnosis strategies are urgently needed for early hcc 
 detection . along these lines , core - fucosylation has recently been 
 used as a potential marker for various cancers , including pancreatic 
 cancer , lung cancer , and liver cancer . afp - l3 , 
 where lens culinaris agglutinin ( lca ) 
 binds to the core - fucosylated glycopeptide of afp , has been used as 
 an alternative marker for hcc . a commercial 
 diagnostic kit for afp - l3 amagasaki ) . using this afp - l3 kit for patients 
 with afp < 20 ng / ml , the auc value between hcc and chronic liver 
 disease was 0.707 , with a specificity of 85.1% at a sensitivity of 
 41.5% . in other work , the aleuria aurantia lectin ( aal)-binding part of alpha-1-antitrypsin 
 ( a1at ) , which comprises both core - fucosylated and antennary - fucosylated 
 glycopeptides , was found to have an auc value of 0.867 to distinguish 
 hcc from cirrhosis samples with a specificity of 86% at a sensitivity 
 of 70% . further analysis showed that antennary - fucosylated a1at gave 
 more frequent false positives , whereas core - fucosylated a1at did not , 
 indicating that only the increase of core - fucosylated a1at is specific 
 for hcc . data available in the 
 literature indicate that ceruloplasmin is 
 upregulated in various lung , pancreatic , colon / rectum , and 
 liver cancers . in previous work , ceruloplasmin 
 was found to be upregulated in lca - enriched hcc serum samples , which 
 indicates the upregulation of core - fucosylated ceruloplasmin . however , it was not known whether the observed increase was common 
 to hcc with various etiologies or unique to hcc with a specific etiology . additionally , ceruloplasmin contains several glycosites , so it is 
 unclear which sites contribute to the effect as well as the relative 
 contribution of each site . in this study , the site - specific 
 core - fucosylation ratio of ceruloplasmin 
 in patients with liver diseases ( cirrhosis or hcc ) of three major 
 etiologies ( alc , hbv , or hcv ) was analyzed using a mass spectrometry - based 
 assay . our study shows that there are four core - fucosylated sites 
 in ceruloplasmin , among which the core - fucosylation ratio of sites 
 138 and 397 are more susceptible to change in disease samples . the 
 most significant change among normal controls , alc - related cirrhosis , 
 and alc - related hcc samples can be attributed to the core - fucosylation 
 ratio of site 138 with an auc value of 0.922 between normal and alc - related 
 cirrhosis and an auc value of 0.838 between alc - related cirrhosis 
 and alc - related hcc , whereas in patients with hbv or hcv etiology , 
 no significant change was observed in cirrhosis samples versus hcc 
 samples . all clinical investigations 
 were conducted 
 according to the principles expressed in the declaration of helsinki . barcelona clinical liver cancer ( bclc ) staging system was used to 
 divide hcc samples into two groups : samples at stage a were considered 
 to be early stage and samples at stage b , c , or d were considered 
 to be late stage . the set of serum samples used in this study 
 contained a total of 116 samples , which were composed of 15 normal 
 controls , 18 alc - related cirrhosis samples , 12 alc - related early stage 
 hcc samples , 12 alc - related late stage hcc samples , 9 hbv - related 
 cirrhosis samples , 6 hbv - related early stage hcc samples , 3 hbv - related 
 late stage hcc samples , 18 hcv - related cirrhosis samples , 9 hcv - related 
 early stage hcc samples , and 14 hcv - related late stage hcc samples . this study was approved by the institutional review board of the university 
 of michigan medical school . in north america , about 3050% 
 of hcc is related to hcv infection and about 10% is related to hbv 
 infection . because of the limited access 
 to hbv - related liver disease samples in the united states , only 9 
 hbv - related cirrhosis and 9 hbv - related hcc samples were included 
 in this study . es , early stage ( bclc staging , stage 
 a ) ; ls , late stage ( bclc staging , stage b , c , or d ) . the procedure 
 was performed following 
 previously described methods with some 
 modifications . human serum 
 ( 10 l ) was thawed on ice and diluted to 200 l with coupling 
 buffer ( 10 mm sodium phosphate , 150 mm sodium chloride , ph 7.2 ) using 
 a cross - link ip kit ( pierce scientific , rockford , il ) . igg is the 
 most abundant glycoprotein in serum , with a concentration of 816 
 mg / ml , and has high affinity to protein a / g agarose beads . igg will 
 severely contaminate the immunoprecipitated target protein , so it 
 was depleted prior to ceruloplasmin immunoprecipitation . in this procedure , diluted serum was incubated with 100 l of protein a / g agarose 
 slurry in a 900 l spin column on an end - to - end rotator at 4 
 c for 3 h. the depleted serum dilution was spun in a centrifuge 
 at 1500 g for 30 s. the beads were washed once with 
 100 l of coupling buffer . immunoprecipitation was performed 
 using the cross - link ip kit according to the supplier s protocol . monoclonal ceruloplasmin antibody ( 3 g ; abcam , cambridge , ma ) 
 was bound to 20 l of protein a / g plus agarose slurry at room 
 temperature for 30 min in 100 l of binding buffer system and 
 cross - linked with the beads by 9 l of 2.5 mm disuccinmidyl 
 suberate ( dss ) cross - linker in 50 l of binding buffer system 
 at room temperature for 30 min . unbound antibody that was not cross - linked 
 was removed by extensive washing with coupling buffer followed by 
 elution buffer ( 100 mm glycine - hcl , ph 2.8 ) . the antibody - conjugated 
 beads were then incubated with igg - depleted serum at 4 c overnight , 
 and elution was carried out with 60 l of elution buffer twice . the eluted ceruloplasmin was neutralized with 12 l of tris 
 saline buffer ( 1 m tris , 0.76 m nacl , 0.052 m kcl , ph 8) to avoid 
 precipitation in the following desalting step . it was then desalted 
 with 500 l zeba micro spin desalting columns 7k mwco ( pierce 
 scientific , rockford , il ) , which were prewashed 5 times with 300 l 
 of 100 mm teab . the desalted sample was dried in a speedvac concentrator 
 ( labconco , kansas city , mo ) at room temperature and redissolved with 
 10 l of 100 mm teab . immunoprecipitated 
 samples were reduced by 20 mm tcep at 37 c for 30 min and were 
 then alkylated by 20 mm iaa at room temperature for 15 min . the samples 
 were then diluted 3 times with 100 mm teab and digested with 0.8 l 
 of trypsin ( 0.5 g/l ) at 37 c overnight . trypsin - digested 
 samples were sonicated in an ice  water bath for 5 min and desalted 
 with a c18 column ( pierce scientific , rockford , il ) . the columns containing 
 8 mg of c18 resin , which can bind up to 30 g of total peptide , 
 were prewetted with 200 l of 0.1% tfa in 50% acetonitrile 5 
 times and equilibrated with 0.1% tfa in water 3 times at 1500 g for 0.5 min . the peptides were bound to the c18 medium 
 5 times followed by 3 times washing with 0.1% tfa to remove nonspecific 
 binding at 1500 g for 0.5 min . twenty microliters 
 of 50% acetonitrile with 0.1% tfa was used for elution at 1500 g for 0.5 min . the elution was repeated twice , and the eluates 
 were pooled for further use . samples were dried in a speedvac concentrator 
 ( labconco , kansas city , mo ) at room temperature . the desalted samples 
 were reconstituted with 29.6 l of endo f3 buffer ( 50 mm sodium 
 acetate , ph 4.5 ) . endo f3 was used to partially truncate the glycan 
 structure of the glycopeptides . after endo f3 digestion , only the 
 innermost glcnac and/or the core - fucose remained attached to the asparagine 
 residue of the peptide , which greatly simplifies the identification 
 of the glycopeptides by mass spectrometry . the samples were pipetted 
 well and sonicated in an ice  water bath for 5 min . endo f3 
 ( 0.4 l , 2 mu ; qa - bio , san mateo , ca ) was added for overnight 
 digestion at 37 c . endo f3 digested samples were deactivated 
 at 95 c for 5 min , and tfa was added to make the final concentration 
 0.1% . after sonication in an ice  water bath for 5 min , the 
 samples were desalted with a c18 column as described above . after 
 evaporation in a speedvac concentrator ( labconco , kansas city , mo ) 
 at room temperature , the samples were ready for mass spectrometry 
 analysis . half of the sample was loaded on an lc - ltq - ms for one run . nano lc - ms / ms conditions were used as 
 described in previous work . in summary , 
 a magic c18 capillary column ( 100 m i.d . 15 cm ; 3 m 
 particles , 200  ) ( michrom biosciences , auburn , ca ) was used 
 for lc separation , and gradient elution was performed using a paradigm 
 mg4 micropump system ( michrom biosciences , auburn , ca ) with a flow 
 rate at 350 nl / min . mobile phase a was 2% acetonitrile with 0.1% formic 
 acid in water , and mobile phase b was 2% water with 0.1% formic acid 
 in acetonitrile . the analytical gradient lasted for 80 min , where 
 the composition of solvent b rose from 5 to 32% in 50 min followed 
 by a washing and equilibration step where solvent b increased to 95% 
 in 1 min , was held for 4 min , returned to 5% b in 0.1 min , and was 
 held for 25 min . an esi - ltq mass spectrometer ( thermo fisher 
 scientific , san jose , ca ) operated in positive ion mode was used for 
 analysis . the esi spray voltage and capillary voltage were set at 
 2.2 kv and 45 v , respectively . collision ionization dissociation ( cid ) 
 fragmentation was performed at 35% of the normalized collision energy . the mass spectra were acquired in a data - dependent manner . following 
 a full scan in the mass range of m / z 4001800 , cid ms / ms was performed on the most intense ions , 
 and the fragment ion in ms / ms with neutral loss of 73 or 48.67 ( m / z ) was selected for cid ms / ms / ms fragmentation . cid ms / ms and ms / ms / ms were performed on the first to the fourth most 
 intense ions from the survey ms full scan . the default charge state 
 was set at 3 + for ms2 and 2 + for ms3 . the scan range of ms2 and ms3 
 is defined automatically by the ltq depending on the default charge 
 states and is dynamic for each ms2 or ms3 scan . the mass spectrum was searched with 
 proteome discoverer 1.2 ( thermo fisher scientific , san jose , ca ) software 
 with sequest using the following settings : ( 1 ) fixed modification : 
 cysteine carbamidomethylation ( + 57.0 da ) ; ( 2 ) dynamic modification : 
 methionine oxidation ( + 16.0 da ) , addition of glcnac ( + 203.1 da ) , or 
 glcnac+fucose ( + 349.2 da ) to asparagine residue ; ( 3 ) one missed cleavage 
 was allowed ; ( 4 ) peptide ion tolerance : 1.4 da ; ( 5 ) fragment ion tolerance : 
 0.8 da ; and ( 6 ) swiss - prot homo sapiens database ( release 2010_10 , downloaded on nov 2 , 2010 ) was used . the identified target peptides were quantified manually with xcalibur 
 qual browser 2.1 ( thermo fisher scientific , san jose , ca ) using the 
 peak area from the extracted ion chromatogram ( xic ) with the following 
 settings : ( 1 ) precursor peaks were extracted with a 1 da ( 0.5 
 da ) mass window , ( 2 ) scan filter was set as full ms , ( 3 ) boxcar - type 
 of smoothing with 7 points was enabled , and ( 4 ) genesis peak detection 
 algorithm was used . the core - fucosylation 
 ratio of each 
 site was calculated as the peak area of core - fucosylated peptides 
 divided by that of non - core - fucosylated peptides . the core - fucosylation 
 ratio of each site in different liver diseases was compared using 
 graphpad prism 5 , and power analysis was done with graphpad statmate 
 2 ( graphpad , la jolla , ca ) . pearson omnibus 
 normality test was used to check the distribution of core - fucosylation 
 ratios of ceruloplasmin at each site before the following student s t - test . the difference between means of the groups was analyzed 
 using the t - test with welch s correction ( gaussian 
 distribution was assumed , and equal standard deviation was not assumed ) 
 at confidence intervals of 95% and two - tailed p values . 
 for power analysis , the average standard deviation ( sd ) of each of 
 the groups being compared was used , with the significance level of 
 0.05 , two - tailed . for example , the core - fucosylation ratio of alc - related 
 cirrhosis at site 138 is 2.170  0.614 ( mean  sd ) and that 
 of alc - related hcc is 3.581  1.424 , so the average sd of the 
 two groups ( 1.019 ) was used for sample size estimation . in order to 
 differentiate the means of the two groups , the sample sizes used in this study guaranteed 
 that all of the significance analyses had statistical power larger 
 than 90% for the observed effect size . the receiver operator characteristic 
 ( roc ) curve of the core - fucosylation ratio between two disease samples 
 or normal control samples was generated with spss 13 ( ibm , armonk , 
 ny ) . the roc analysis of core - fucosylation ratio and afp was performed 
 as follows : ( 1 ) predicted value of probabilities was generated with 
 binary logistic regression using core - fucosylation ratio and afp as 
 covariates and ( 2 ) the roc curve was generated on the basis of the 
 predicted probabilities . the auc accuracy for the roc curve of this 
 combined analysis was checked by 10-fold cross - validation considering 
 the relatively small sample size . it has been shown that the pathology of liver diseases with different 
 etiologies , including alc , hbv , and hcv , is very different . combining all cirrhosis or hcc samples with different etiologies 
 together for analysis may mask potential markers for each type of 
 liver disease . in this work , we have studied the core - fucosylation 
 of serum ceruloplasmin as a marker of hcc , where the study focused 
 specific investigation on each disease subtype . the selection of ceruloplasmin 
 is based on a previous study where using 
 mass spectrometry and lectin blots it was found that some ceruloplasmin 
 proteins that could bind to lca were upregulated in hcc samples compared 
 to those in cirrhosis samples . however , in that study , hcc with various 
 etiologies was not analyzed separately . furthermore , the overall result 
 with lectin blots only revealed that the core - fucosylation ratio of 
 the entire protein was changed without any indication of the possible 
 contribution of each specific site and thus it may mask the core - fucosylation 
 change at the site with the strongest response in certain disease 
 states . in this study , we have identified four core - fucosylated 
 sites of 
 ceruloplasmin and further analyzed the site - specific core - fucosylation 
 ratio changes in liver cirrhosis and hcc with various etiologies , 
 including alc , hbv , and hcv , aiming to search for specific changes 
 that can be used as markers of hcc . ceruloplasmin 
 isolated from the serum sample was first digested into peptides with 
 trypsin and was then digested with endo f3 . the sequence coverage 
 of ceruloplasmin identified by ms was over 50% , as shown in supporting information figure s2 . the sds - page 
 gel result of immunoprecipitated ceruloplasmin from two samples is 
 shown in figure s3 , using 0.3 g 
 of commercially purified ceruloplasmin as control . after endo 
 f3 digestion , only the innermost glcnac and/or the core - fucose remained 
 attached to the asparagine residue of the peptide . this is shown in figure 1a , where 
 the peptide ehegaiypdnttdfqr with core - fucosylated site 138 had a 
 mass of 748.46 , with the charge of 2 + and 3 + . using ms / ms cid in the 
 ltq mass spectrometer , the core - fucose tended to be lost and to generate 
 a neutral loss peak with a mass of m / z 699.8 , a reduction of 146 da ( corresponding to the mass of fucose ) 
 from the parent ion . ms / ms / ms was used sequentially to fragment this 
 neutral loss peak for peptide identification , as shown in figure 1b . for peptides without core - fucosylation , they 
 were readily fragmented in ms / ms . ( a ) ms / ms mass spectrum of the interested 
 core - fucosylated parent 
 ion peptide ehegaiypdnttdfqr at m / z 748.46 with the neutral loss peak ion at m / z 699.8 . ( b ) ms / ms / ms mass spectrum of the neutral loss 
 peak ion from panel a. nl : peak intensity . according to the nxt / s motif ( x is not proline ) for n - glycosylation , 
 there are seven potentially glycosylated sites in ceruloplasmin ( sites 
 138 , 227 , 358 , 397 , 588 , 762 , and 926 ) . sites 227 and 926 were not 
 detected in this experiment and have not been reported in previously 
 published work . sites 138 , 358 , 397 , 
 588 , and 762 were detected in this experiment , but only sites 138 , 
 358 , 397 , and 762 were glycosylated and/or core - fucosylated , as shown 
 in table 2 . all four target core - fucosylated 
 peptides were identified with the same method as described above . the ms / ms and ms / ms / ms spectra and the matched peak list of ms / ms / ms 
 spectra with respective theoretical peaks are shown in supporting information figure s4 . coref : core - fucosylation . observed m / z is 
 calculated as the mean  sd of all identified 
 glycopeptide spectra from five randomly chosen samples . charge 2 + and/or charge 3 + were 
 detected in the target glycopeptides . however , only one of the four target glycopeptides ( site 397 ) has 
 two charge states in all of the tested samples . the work of stavenhagen has shown that the intensities of one peptide 
 with different charge states are different . we have also noticed this 
 from our ltq mass spectrometry result . in this study , the charge state 
 with a stronger peak intensity that could be detected in all tested 
 samples was chosen for each target peptide , as listed in table 2 , to determine the roc value for distinguishing 
 hcc from cirrhosis . the intensity of the core - fucosylated peptide 
 and the non - core - fucosylated peptide in ms1 was integrated , as shown 
 in figure s5 , and the core - fucosylation 
 ratio was calculated as follows : from figure s5 , it was also found that 
 each core - fucosylated peptide eluted about 
 30 s before its related non - core - fucosylated peptide . this is probably 
 due to the addition of core - fucose , which reduces its hydrophobicity . the potential of false positives during integration is avoided 
 by ( 1 ) immunoprecipitation , which simplifies the sample complexity 
 analyzed by the ltq , and ( 2 ) a careful check of the spectra of the 
 target peptides based on the retention time , the m / z of the parent ion , and the ms2 and ms3 fragmentation 
 profiles . note that the intensity of these glycopeptides does 
 not reflect the exact quantity of the peptides because of their different 
 ionization efficiencies . this index was 
 used to make a comparison between different disease states rather 
 than to provide the ratio of the exact quantity of the glycopeptides . each 
 core - fucosylated site of ceruloplasmin appears to have a different 
 response in hcc samples compared to that in normal controls . the core - fucosylation 
 ratio in alc - related hcc early stage and late stage samples was significantly 
 higher than those of normal controls at all four sites ( figures 2 and 3 ) , whereas in hbv - related 
 hcc samples , the upregulation of core - fucosylation was found at site 
 397 ( figure 4 ) and at sites 138 and 397 in 
 hcv - related hcc samples ( figure 5 ) . thus , all 
 four sites in ceruloplasmin are sensitive to alc - related hcc , whereas 
 in hbv / hcv - related hcc samples , the core - fucosylation ratio at site 
 138 and/or site 397 are more susceptible to change , whereas sites 
 358 and 762 are less responsive . core - fucosylation ratios of ceruloplasmin 
 at sites 138 ( a ) , 397 
 ( b ) , 762 ( c ) , and 358 ( d ) in normal and alc - related serum samples . hcce , hcc early stage ; hccl , hcc late stage ; hcc , all hcc samples . a t - test comparison was made between each disease 
 group and normal group as well as between cirrhosis and each disease 
 group . error bar indicates sem . * , p < 0.05 ; * * , p < 0.01 ; * * * , p < 0.001 . roc analysis of the core - fucosylation ratio at site 138 
 between 
 alc - related hcc and cirrhosis samples ( a ) and between alc - related 
 hcc and normal samples ( b ) . ( c ) combination analysis of afp and the 
 core - fucosylation ratio at site 138 between alc - related cirrhosis 
 and hcc samples . ( d ) two - dimensional plot of afp and core - fucosylation 
 ratio at site 138 between alc - related cirrhosis and hcc samples . core - fucosylation ratios of ceruloplasmin at 
 sites 138 ( a ) , 397 
 ( b ) , 762 ( c ) , and 358 ( d ) in normal and hbv - related serum samples . a t - test comparison was made between each disease 
 group and normal group as well as between cirrhosis and each disease 
 group . error bar indicates sem . * , p < 0.05 ; * * , p < 0.01 ; * * * , p < 0.001 . core - fucosylation ratios of ceruloplasmin at sites 138 
 ( a ) , 397 
 ( b ) , 762 ( c ) , and 358 ( d ) in normal and hcv - related serum samples . hcce , hcc early stage ; hccl , hcc late stage ; hcc , all hcc samples . a t - test comparison was made between each disease 
 group and normal group as well as between cirrhosis and each disease 
 group . error bar indicates sem . * , p < 0.05 ; * * , p < 0.01 ; * * * , p < 0.001 . note that the four different glycopeptides have 
 different core - fucosylation 
 ratios using our index formula ( figure 2 ) . this index does not indicate the actual absolute quantity of the core - fucosylation 
 level of those sites . one issue is that trypsin cleavage may be inhibited 
 by the nearby core - fucosylation . among 
 the four targeted core - fucosylation sites , three sites do not have 
 this problem , whereas site 397 may inhibit trypsin digestion at its 
 n side . although no missed cleavage was detected , it is difficult 
 to know if there is a missed cleavage . if there is one missed cleavage 
 before site 397 , the peptide will contain 43 amino acids , which is 
 beyond the detection limit of our mass spectrometer . therefore , the 
 core - fucosylation ratio calculated for site 397 does not indicate 
 the actual core - fucosylation level with full digestion . also , previous 
 studies have shown that different glycopeptides may have different 
 ionization efficiencies in ms . equal 
 molar amounts of peptides and glycopeptides with the same peptide 
 backbone have different signal intensities in ms . therefore , the core - fucosylation ratio calculated in this 
 study does not provide the absolute quantity of the core - fucosylation 
 level of those sites . nevertheless , all samples in this study were 
 processed following the same procedure so that the level of digestion 
 should be similar among samples , and the response in ms of the same 
 core - fucosylated peptide should be the same among samples . thus , even 
 when we do not obtain the true ratio , we are still able to use this 
 index to compare different disease states , which is the object of 
 this work . the core - fucosylation changes 
 of ceruloplasmin at different sites were distinctly different in various 
 liver diseases . the core - fucosylation ratio changed dramatically in 
 alc - related hcc samples but not in hbv- or hcv - related hcc samples 
 ( figures 2 , 4 and 5 ) . compared to that of normal samples , the 
 core - fucosylation ratio of ceruloplasmin in alc - related hcc samples 
 was significantly upregulated at all four sites , with p < 0.001 . when comparing alc - related cirrhosis with alc - related 
 hcc samples , the core - fucosylation ratio of three sites was upregulated 
 in hcc samples , with p < 0.001 at site 138 and p < 0.01 at sites 397 and 762 the level of change was 
 not significantly different for early stage or late stage hcc samples . the roc curve analysis between alc - related hcc 
 samples and normal samples at site 138 resulted in an auc value of 
 0.922 with a specificity of 80.0% at a sensitivity of 87.5% ( figure 3a ) . the roc curve analysis at site 138 between alc - related 
 cirrhosis and hcc resulted in an auc value of 0.838 with a specificity 
 of 77.8% at a sensitivity of 79.2% ( figure 3b ) . when combining the core - fucosylation ratio of site 138 and the 
 clinical afp value , as shown in figure 3c , 
 the auc value between alc - related cirrhosis and hcc increased to 0.954 
 ( confirmed by 10-fold cross - validation ) ( orsite138 = 12.26 , p = 0.017 ; orafp = 3.64 , p = 
 0.022 ) . the model fit was significantly improved over the one with 
 afp alone ( auc value = 0.867 ) ( lr test p = 0.0002 ) . this improvement is mainly due to the enhanced sensitivity from 78.2 
 to 91.3% , as shown in 2d plot ( figure 3d ) . 
 here , the optimum sensitivity and specificity of afp was used instead 
 of that at the 20 ng / ml cutoff that is widely used in clinical practice . the reason is that only 25% of alc - related hcc samples used in this 
 study have afp > 20 ng / ml . therefore , in alc - related patients , 
 the 
 afp cutoff should be a lower value than 20 ng / ml . the core - fucosylation ratio of ceruloplasmin at site 138 could be 
 a good complementary biomarker of afp for screening in this population . the roc curve analysis of the other three sites showed lower auc values 
 than that at site 138 ( supporting information 
 figure s6 ) . in this study , the charge state with stronger 
 peak intensity that 
 could be detected in all tested samples was chosen for each target 
 peptide . only one of the four target glycopeptides ( site 397 ) has 
 two charge states in all of the tested samples . the core - fucosylation 
 ratio of charge 2 + peptides with asn397 has been listed in this article , 
 which has a stronger peak intensity . the core - fucosylation ratio of the two charge states turned out 
 to be somewhat different in our study ( p < 0.05 ) , 
 where charge 2 + has an roc value of 0.723 and charge 3 + has an roc 
 value of 0.813 to distinguish alc - related cirrhosis and alc - related 
 hcc , although the correlation of the two is 0.921 . the 
 roc value of the core - fucosylation ratio of one peptide to distinguish 
 two disease states is charge - state - specific . two other clinical 
 indices , inr and hgb , also showed enhanced auc 
 values at 0.877 and 0.880 , respectively , when combined with the core - fucosylation 
 ratio at site 138 to distinguish alc - related hcc from alc - related 
 cirrhosis samples ( supporting information figure 
 s8 ) . inr stands for international normalized ratio , a measure 
 of blood coagulation that is based on prothrombin time , and hgb stands 
 for the amount of hemoglobin in blood . in hbv - related samples , the core - fucosylation ratio of hcc samples 
 at site 397 was higher than that in normal samples , with p < 0.05 ( figure 4 ) , whereas hcv - related 
 hcc samples had significant upregulation of core - fucosylation at sites 
 138 and 397 , with p < 0.05 and p < 0.001 , respectively ( figure 5 ) . however , 
 a statistically significant core - fucosylation change was not found 
 between hbv - related cirrhosis and hcc samples or between hcv - related 
 cirrhosis and either early stage hcc or late stage hcc samples . samples with the same state regardless 
 of their etiologies were pooled for further analysis ( figure 6 ) , which included 45 cirrhosis samples , 56 hcc samples , 
 and 15 normal samples . the statistical result showed that the core - fucosylation 
 ratios at all four sites were higher in hcc samples than those in 
 normal samples ( p < 0.01 ) ( figure 6a ) . meanwhile , despite the remarkable difference of the core - fucosylation 
 ratio at site 138 in alc - related cirrhosis and hcc samples ( auc = 
 0.838 , p < 0.001 ) , after mixing samples of all 
 etiologies the core - fucosylation ratio at site 138 appeared to have 
 a much lower significance level ( auc = 0.583 , p = 
 0.034 ) between cirrhosis and hcc samples because of the low values 
 in hbv - related samples ( auc = 0.519 ) and in hcv - related samples ( auc 
 = 0.666 ) ( figure 6b ) . clinicians usually follow specific etiologies , generally 
 hcv and alcohol related in the u.s . if the etiology is unknown ( i.e. , 
 whether the patient had hcv or alcohol related ) , then it might be 
 of limited value given our results on the mixed samples . however , 
 we would expect to see an effect on this site for an unknown that 
 was alcohol related , whereas with hcv we would expect to see a minimal 
 response . ( a ) core - fucosylation ratios of ceruloplasmin at sites 138 , 358 , 
 397 , and 762 in normal samples and cirrhosis and hcc samples with 
 mixed etiologies . a t - test comparison was made between 
 each disease group and normal group as well as between cirrhosis and 
 each disease group . error bar indicates sem . * , p < 0.05 ; * * , p < 0.01 ; * * * , p < 0.001 . ( b ) receiver operating curve ( roc ) analysis of the core - fucosylation 
 ratio at site 138 in cirrhosis and hcc samples with a single etiology 
 and with mixed etiologies . in terms of marker discovery , it is thus necessary to separate 
 hcc samples with different etiologies to avoid masking potential changes 
 that could serve as markers . the site - specific analysis of the core - fucosylation 
 ratio of ceruloplasmin provides a potentially important marker for 
 early hcc detection in cirrhosis patients with alc etiology . samples with the same etiology ( alc / hbv / hcv ) regardless of the 
 disease states were also pooled and analyzed ( supporting information figure s9 ) . alcohol intake and hbv 
 or hcv infection all increased core - fucosylation ratios of ceruloplasmin 
 at most of the sites with different significance levels compared to 
 normal samples . other potential markers for hcc were also studied 
 for site - specific 
 core - fucosylation changes , including alpha-2 macroglobulin and transferrin , 
 with similar procedures described above . neither of these proteins 
 showed a significant difference in the level of core - fucosylation 
 at any of the potential glycosites between cirrhosis and hcc samples 
 with either alc , hbv , or hcv etiology ( data not shown ) . using a mass - selected site - specific assay , we have found that altered 
 site - specific core - fucosylation of ceruloplasmin can serve as a potential 
 marker for alc - related hcc . site 138 of ceruloplasmin , compared with 
 other core - fucosylated sites , is the most highly core - fucosylated 
 and is one of the sites that is most susceptible to change . this site 
 showed the highest auc value of 0.838 to distinguish alc - related hcc 
 from alc - related cirrhosis samples . when combining the core - fucosylation 
 ratio of site 138 and the level of afp , the auc value increased to 
 0.954 , which was markedly improved compared to that of afp alone ( auc 
 = 0.867 ) . however , in hbv- or hcv - related disease samples , although 
 the core - fucosylation ratio at sites 138 and/or 397 was significantly 
 higher than that in normal samples , no difference was found between 
 cirrhosis and hcc samples . when combining samples with the same disease 
 states regardless of their etiologies , the core - fucosylation ratio 
 at site 138 showed a much lower auc level ( 0.583 ) to distinguish between 
 cirrhosis and hcc samples compared with that in alc - related disease 
 samples ( auc = 0.838 ) . therefore , the core - fucosylation ratio of ceruloplasmin 
 at site 138 may serve as a potential marker for alc - related hcc , which 
 is both site - specific and etiology - specific . our findings reveal a new field 
 for future marker discovery : the core - fucosylation change of a specific 
 glycosite rather than of the entire protein may serve as an improved 
 marker for disease diagnosis ; meanwhile , markers targeting etiology - specific 
 hcc may improve the specificity and sensitivity .
a mass 
 spectrometry - based methodology has been developed to study 
 changes in core - fucosylation of serum ceruloplasmin that are site - specific 
 between cirrhosis and hepatocellular carcinoma ( hcc ) . 
 the serum samples 
 studied for these changes were from patients affected by cirrhosis 
 or hcc with different etiologies , including alcohol , hepatitis b virus , 
 or hepatitis c virus . 
 the methods involved trypsin digestion of ceruloplasmin 
 into peptides followed by endo f3 digestion , which removed most of 
 the glycan structure while retaining the innermost n - acetylglucosamine ( glcnac ) and/or core - fucose bound to the peptide . 
 this procedure simplified the structures for further analysis by mass 
 spectrometry , where four core - fucosylated sites ( sites 138 , 358 , 397 , 
 and 762 ) were detected in ceruloplasmin . 
 the core - fucosylation ratio 
 of three of these sites increased significantly in alcohol - related 
 hcc samples ( sample size = 24 ) compared to that in alcohol - related 
 cirrhosis samples ( sample size = 18 ) , with the highest auc value of 
 0.838 at site 138 . when combining the core - fucosylation ratio of site 
 138 in ceruloplasmin and the alpha - fetoprotein ( afp ) value , the auc 
 value increased to 0.954 ( orsite138 = 12.26 , p = 0.017 ; orafp = 3.64 , p = 0.022 ) , which 
 was markedly improved compared to that of afp ( auc = 0.867 ) ( lr test p = 0.0002 ) alone . 
 however , in hbv- or hcv - related liver 
 diseases , no significant site - specific change in core - fucosylation 
 of ceruloplasmin was observed between hcc and cirrhosis .