kidney 
 ( renal ) cancer , 
 which includes renal cell carcinomas and 
 transitional cell carcinomas of the renal pelvis , is estimated to 
 have caused 13  680 deaths ( 8780 men and 4900 women ) in 2013 . 

clear cell renal cell carcinoma ( ccrcc ) is the most common subtype 
 of kidney cancer , contributing approximately 80% of all reported cases , 
 whereas the papillary and chromophobe subtypes contribute 15 and 5% , 
 respectively .
currently , there are 
 no biomarkers for ccrcc early diagnosis , and 
 the standard method of treatment ( i.e. , surgery ) is unsatisfactory . 
 this is because patients undergoing surgery are likely to relapse 
 at a rate of 2050% and have a higher chance of developing 
 metastatic tumor , which is incurable .
therefore , 
 there is the urgent need to identify biomarkers for early detection 
 and to predict or monitor the recurrence of ccrcc after surgery .
the complexity ( dynamic range of approximately 10 ) 
 of the plasma proteome is of great concern in biomarker discovery 
 studies .
this is because tissue and secreted cell surface protein 
 products , which are indicators of a disease and/or healthy state , 
 are of low abundance in blood plasma .
for example , current clinical 
 biomarkers including prostate specific antigen ( psa ) and her2/neu 
 are present at low nanograms per milliliter concentration in plasma , and the ability to identify such molecular markers 
 requires a comprehensive multi - dimensional analytical approach .
this approach is sensitive and specific because it involves 
 the characterization of a subpopulation of proteins whose alterations 
 are associated with many diseases , including cancer .
protein glycosylation is one of the most diverse and frequently 
 occurring post - translational modifications involved in a number of 
 cell processes , and aberrant changes in 
 glycosylation profile during the development and progression of cancer 
 is known .
another method is immuno - affinity depletion , which targets 
 the removal of high - abundance proteins , enabling a deeper mining of 
 low - level proteins .
these strategies have been used either alone or 
 in combination to enhance protein biomarker discovery studies .
a number of groups have reported the use of high - abundance proteins 
 depletion and enrichment of target glycoproteins and have observed 
 differential protein expression and alterations in protein glycosylation 
 in cancer samples .
these observations suggest that the integration of protein depletion 
 and enrichment of target glycoproteins enhance proteomics and glycoproteomics 
 studies , leading to the identification of potential candidate markers .
ccrcc plasma biomarker discovery is lagging behind other disease 
 studies , although early diagnosis of ccrcc can lead to a cure by surgery . 

to date , only a handful of ccrcc plasma proteomics biomarker discovery 
 studies have been reported , and there are not yet 
 any reports on n - glycan profiles in the literature to the best of 
 our knowledge . therefore , we attempted to use a comprehensive comparative 
 omics approach , namely , proteomics , glycoproteomics , and n - glycomics , 
 to study ccrcc plasma before ( disease ) and after ( non - disease ) curative 
 nephrectomy , and we evaluated the alterations associated with histological 
 status .
our goal was to identify potential candidate markers of biological 
 interest in ccrcc development and their potential utility to monitor 
 ccrcc recurrence after curative nephrectomy .
our laboratory 
 previously developed an automated high - throughput 
 multi - lectin affinity chromatography ( hp - m - lac ) platform that combines 
 two high - abundance proteins ( albumin and igg ) depletion and multi - lectins 
 ( con a , wga , and jac ) fractionation . in 
 the current study , we have expanded the hp - m - lac approach to incorporate 
 12 high - abundance proteins depletion ( 12p ) and multi - lectins ( sambucus nigra ( sna ) , aleuria aurantia ( aal ) , and phytohemagglutinin - l ( pha - l ) ) enrichment with the overall 
 goal of improving the proteomic and/or glycoproteomic depth .
aal , 
 sna , and pha - l lectins have specificities toward core ( 1,6 ) 
 fucose , sialic acid , and n - acetyl glucosamine as well as highly 
 branched glycans of terminal galactose and mannose oligosaccharides .
these lectins were selected to capture 
 the population of glycan structures frequently implicated in cancer 
 progression and metastasis .
ms / ms analysis of trypsin - digested , 12p - depleted 
 plasma and m - lac fractions ( bound and unbound ) enabled relative quantification 
 via spectral counting .
furthermore , n - glycans 
 released from m - lac fractions were profiled , and detailed structural 
 annotation was conducted .
we identified several low - abundance proteins 
 and glycoproteins with significant differential abundance levels in 
 ccrcc cancer samples .
glycosylation alterations in cancer plasma ( before ) 
 compared to noncancer plasma ( after ) were evident on the basis of 
 glycoproteins differential binding to the m - lac column and n - glycan 
 profiles .
capture select 12p depletion 
 resin and peek columns were purchased from life technology ( milford , 
 ma ) . gravity omnifit glass column was provided by biochem fluidics 
 ( boonton , nj ) .
poros r1 50 m bulk media ( reversed - phase packing ) 
 and hplc self - packing device were purchased from applied biosystems 
 ( framingham , ma ) .
bis - tris sodium dodecyl sulfate polyacrylamide 
 gel electrophoresis ( sds - page ) gels ( 412% ) and nupage mes 
 sds running buffer ( 10 ) were purchased from invitrogen ( carlsbad , 
 ca ) .
hplc - ms grade water , formic acid , acetonitrile , and other buffer 
 reagents were all purchased from thermo fisher scientific ( waltham , 
 ma ) .
clear cell renal 
 cell carcinoma ( ccrcc ) patients enrolled in this study gave their 
 consent via protocols 01 - 130 approved by the institutional review 
 board at massachusetts general hospital and provided to us by dr . 

twenty ccrcc plasma samples before nephrectomy and 20 ccrcc plasma 
 samples after nephrectomy were pooled to give one disease ( rcc ( + ) ) 
 and one non - disease ( rcc (  ) ) sample .
pooling was necessary 
 for this discovery study due to the limited amount of samples and 
 also to reduce variability among patients .
pooled plasma samples were 
 stored in 80 c and did not undergo more than two freeze / thaw 
 cycles . an automated hplc platform used for 
 high - abundance protein depletion and glycoprotein fractionation
has 
 been described previously , and we have 
 applied this fractionation platform with moderate changes . briefly , 
 pooled plasma samples were depleted using a 12 ( albumin , igg , igm , 
 iga , free light chains , fibrinogen , transferrin , 1 antitrypsin , 
 apolipoprotein a1 , 2 macroglobulin , haptoglobin , and 1 
 acid glycoprotein ) abundance protein depletion column packed in - house 
 into a peek column ( 4.6 mm  100 mm ) followed by glycoprotein 
 fractionation with a multi - lectin affinity column ( m - lac ) containing 
 equal mixtures of lectins : aleuria aurantia lectin ( aal ) , sambucus nigra lectin 
 ( sna ) , and phaseolus vulgaris leucoagglutinin 
 ( pha - l ) .
three columns ( 12p , m - lac , and r1 reversed phase ) , each attached to 
 a separate valve , were connected in series on a two - dimensional hplc 
 system ( shimadzu , columbia , md ) equipped with an on / off switch to 
 control the valves . during sample fractionation ,
the columns were 
 first equilibrated with a binding buffer ( 25 mm tris , 0.5 m sodium 
 chloride , 1 mm mncl2 , 1 mm cacl2 , and 0.05% 
 sodium azide , ph 7.4 ) at a flow rate of 2.0 ml / min for 15 min followed 
 by plasma loading at a flow rate of 0.5 ml / min for 25 min .
depleted 
 plasma ( 12p unbound ) , m - lac bound , and m - lac unbound fractions were 
 eluted separately via valve switching and desalted on an r1 reversed - phase 
 column using a 70% solvent b ( 0.1% trifluoroacetic acid in acetonitrile ) 
 and 30% solvent a ( 0.1% trifluoroacetic acid in milli - q water ) gradient . 

elution buffers for 12p depletion and m - lac fractions were 0.1 m glycine 
 ( ph 2.5 ) and 0.1 m acetic acid ( ph 2.5 ) , respectively .
total protein 
 concentration measurements of all collected fractions were performed 
 using qubit fluorescence assay ( life technologies , inc . ,
briefly , 20 g 
 of total protein per sample ( 2 analytical replicates of each fraction ) 
 was brought to 100 l volume with ultrapure water and 9 volumes 
 of acetone added followed by overnight precipitation at 20 
 c .
precipitated proteins were centrifuged briefly , acetone was 
 removed , and the samples were speed vacuumed to dryness followed by 
 resolubilization of the protein pellet in 10 l urea ( 8 m ) . 

the protein solution was dot blotted onto a 100% ( v / v ) methanol - activated 
 pvdf membrane ( millipore ) surface and dried at room temperature .
protein 
 spots were visualized using direct blue 71 ( sigma - aldrich ) and destained 
 with 40% ( v / v ) ethanol and 10% ( v / v ) acetic acid .
the membrane 
 was then blocked with 1% ( w / v ) pvp40 solution followed by 3 washes 
 of 5 min each with water .
pngase f ( 2.5 u ; flavobacterium 
 meningospeticum , roche ) was added , and the mixture 
 was incubated at 37 c for 15 min and further incubated at 37 
 c overnight after an additional 10 l of water was added . 

n - glycans were extracted in the following fashion : 5 min sonication 
 of the 96-well plate containing glycans and three washes with 20 l 
 of water .
samples were acidified with 10 l 
 of 100 mm ammonium acetate ( ph 5 ) and incubated at room temperature 
 for 1 h. samples were subsequently dried via speed vacuum and reduced 
 with 20 l of 1 m nabh4 in 50 mm koh at 50 c 
 for 3 h. the reaction was stopped by addition of 2 l of acetic 
 acid and desalted using ag 50w x8 cation exchange resin ( bio - rad ) . 

methanol was added to remove any residual borate and allowed to evaporate 
 in the vacuum centrifuge .
separation of the n - glycan 
 alditols was performed using a hypercarb pgc ( 5 m hypercarb , 
 180   100 mm ; thermo fisher scientific ) connected on 
 the hplc system ( agilent 1100 ) over an 85 min gradient from 0 to 45% 
 acetonitrile in 10 mm ammonium bicarbonate , and eluted
ms / ms on a agilent msd three - dimensional 
 ion - trap xct plus mass spectrometer .
settings for the ms / ms were as 
 follows : drying gas flow , 7 l / min ; drying gas temperature , 325 c ; 
 nebulizer gas , 18 psi ; skimmer , 40.0 v ; trap drive , 99.1 
 v ; and capillary exit , 166 v. smart fragmentation was used 
 with starting and ending amplitudes of 30 and 200 , respectively .
ions 
 were detected in ion charge control targeted at 100  000 ions 
 with a maximum accumulation time of 200 ms .
ms spectra were obtained 
 in negative ion mode with two scan events : a full scan range between m / z 100 and 2200 at a scan speed of 8100 m / z / s and a dependent ms / ms scan after 
 cid of the top two most intense precursor ions with threshold 30  000 
 and relative threshold of 5% base peak .
mass accuracy calibration 
 of instrument was performed using tuning mix ( agilent ) , and n - glycans 
 released from bovine fetuin served as positive controls before each 
 data set run .
twenty micrograms of total protein from the depleted 
 plasma and m - lac fractions was resolved on a 412% bis - tris 
 sds - page gel ( novex nupage , life technologies ) followed by trypsin 
 ( promega , madison , wi ) digestion of the excised gel pieces as previously 
 described .
briefly , lanes were cut into 
 four bands , and each band was cut into 1  1 mm pieces . 

gel pieces were trypsin ( 0.04 g/l ) digested following 
 destaining with 50 mm ammonium bicarbonate buffer at ph 8.0 and acetonitrile , 
 reduction ( 25 mm dithiothreitol ) at 56 c for 30 min , and alkylation 
 ( 50 mm iodoacetamide ) at room temperature for 30 min in darkness . 

trypsin digests were extracted with 100 l of 50% ( v / v ) acetonitrile/0.1% 
 ( v / v ) formic acid in hplc grade water three times and speed vacuumed 
 to dryness .
mass spectrometry analysis of depleted plasma and m - lac 
 fractions was performed on an ltq - orbitrap elite instrument ( thermo 
 fisher scientific , waltham , ma ) equipped with an ultimate 3000 hplc 
 ( lc packings - dionex , marlton , nj ) and nano - esi source . a reversed - phase 
 c18 column packed in - house with a 75 m metal spray tip (
peptides were separated at a flow 
 rate of 200 nl / min on the c18 column using the following 100 min gradient : 
 540% buffer b for 80 min , 4090% buffer b for 15 min , 
 and 902% buffer b for 5 min . mobile phase a consisted of 0.1% 
 v / v formic acid in hplc grade water , and mobile phase b consisted 
 of 0.1% v / v formic acid in acetonitrile .
the mass spectrometer was 
 operated in a data - dependent mode , with the 8 most abundant precursor 
 ions selected for collision induced dissociation ( cid ) ms / ms fragmentation 
 in a full ms scan range of m / z 4002000 
 with a mass resolution of 120  000 .
dynamic exclusion parameters 
 were set to 1 repeat count ( repeat duration , 30 s ; exclusion list 
 size , 100 ; exclusion duration , 45 s ; and exclusion mass width , 1.0 m / z low and 1.50 m / z high ) .
analysis of n - glycan data was performed using esi - compass 
 1.3 ( bruker 
 daltonics ) .
monoisotopic masses obtained were searched against glycomod 
 ( http://web.expasy.org/glycomod/ ) for possible glycan compositions 
 and subsequently verified by their corresponding ms / ms spectra .
the 
 relative abundance of each glycan in a sample was determined using 
 the peak area of each glycan against the sum of peak areas of all 
 glycans from extracted ion chromatograms , which has been shown to 
 be a reasonably accurate method for relative n - glycan quantitation .
lc  ms / ms proteomic and glycoproteomic 
 data were searched against an annotated human database ( release 2013_1 ; 
 34  157 entries ) using the sequest algorithm ( thermo electron 
 corp , san jose , ca ) in the thermo fisher proteome discoverer 1.4 suite . 

peptide identification was based on the hupo criteria , which included 
 cn  0.1 , peptide probability < 0.001 , and xcorr 
  1.9 , 2.5 , and 3.8 for singly , doubly , and triply charged 
 ions , respectively .
confidence in identification was further increased 
 by applying the reverse database with a false discovery rate ( fdr ) 
 targeted at 1% at the peptide level .
other search parameters included 
 a maximum of 2 missed cleavages , full trypsin as enzyme , carbamidomethylation 
 on cysteine as a static modification , and deamidation of asparagine 
 as a dynamic modification ; precursor ion mass tolerance and fragment 
 ion mass tolerance were set at 5 ppm and 0.8 da , respectively .
panther 
 ( protein analysis through evolutionary relationships ) database ( http://pantherdb.org/ ) was used for gene ontology classification . 

a label free semiquantitative method using spectral count was applied 
 to select proteins of interest with abundance changes and was used 
 to evaluate m - lac differential binding in rcc samples as previously 
 described .
briefly , ratios of spectral 
 count in disease to spectral count in non - disease allowed us to select 
 proteins with potential abundance level changes after normalization 
 with a reference ratio calculated from total spectral counts .
glycoprotein 
 candidates with m - lac differential binding were selected on the basis 
 of the ratios of the spectral count of the m - lac bound fraction considered 
 theoretical ( non - disease_bound measured  ( disease_unbound measured / non - disease_unbound 
 measured ) ) to the spectral counts of the experimental m - lac bound 
 fraction ( disease_bound ) . in instances
where no peptides ( 0 ) were 
 observed for a particular protein under consideration , 1 was added 
 for meaningful ratio calculations .
proteins or glycoproteins with 
 a fold change  3 or  3 were identified as differentially 
 expressed .
excel software ( microsoft office 2010 ) was used to generate p - values by calculating standard student s t - test and to investigate n - glycome and n - glycoproteome 
 differential expression by considering p - values  
 0.05 as statistically significant .
the global profiles 
 of the proteome , glycoproteome , and n - glycome of ccrcc plasma were 
 achieved by designing an analytical strategy that focused on deeper 
 mining of low - abundance disease - associated nonglycoproteins , glycoproteins , 
 and n - glycans ( figure 1 ) . in our previous publications
, 
 we have shown that a multi - dimensional platform is a valuable approach 
 to comprehensively characterize the proteome and glycoproteome of 
 biological samples to enhance the identification of potential biomarker 
 candidates present at low amounts . in the current study ,
plasma samples were initially 
 purified to reduce the large dynamic range of plasma concentration 
 by depleting the top 12 high - abundance proteins ( 12p ) .
furthermore , 
 a semitargeted approach was used as a second fractionation strategy , 
 in which equal mixtures of three lectins , aleuria aurantia ( aal ) , sambucus nigra ( sna ) , and phaseolus vulgaris leucoagglutinin ( pha - l ) , were 
 packed into an hplc peek column to enrich the subglycoproteome .
lectins 
 have previously been used to target glycan structures commonly altered 
 in carcinogenesis . in a serum breast cancer proteomic study , zeng et al . observed that 
 differential protein affinities toward selected lectins was indicative 
 of changes in glycan expression level in cancer versus control samples .
used pha - l lectin to 
 capture potential breast carcinoma biomarkers elevated in breast carcinoma 
 tissues at different stages .
experimental 
 workflow showing the process used in the characterization 
 of clear cell renal cell carcinoma plasma ( ccrcc ) .
ccrcc plasma samples 
 were depleted of the top 12 most highly abundant proteins followed 
 by glycoproteins enrichment and lc  ms / ms analysis of depleted 
 plasma and m - lac of bound and unbound fractions . in this study , we focused on 40 plasma samples obtained from 
 20 
 patients diagnosed with clear cell renal cell carcinoma ( supporting information table s1 ) .
plasma samples 
 taken before ( rcc ( + ) ) and after ( rcc (  ) ) curative nephrectomy 
 were pooled into two groups : disease ( before nephrectomy , n = 20 ) and non - disease ( after nephrectomy , n = 20 ) .
pooling was necessary to reduce patient variability and to improve the effective depletion of plasma 
 while increasing protein detection coverage as previously established .
also , pooling allowed for a sufficient amount 
 of samples to be available for two analytical replicates of each omic 
 analysis .
12p - depleted plasma and m - lac fractions were subjected to 
 proteomics , glycoproteomics , and n - glycomic analysis using analytical 
 platforms described in the materials and methods .
advances in proteomic analysis have pointed out the importance of 
 minimizing interference from high - abundance proteins that may mask 
 and/or prevent the detection of low - level proteins in disease samples .
therefore , an analytical technology that improves the identification 
 of low - level proteins and increases the depth of proteomic data is 
 desirable . in the current study , we utilized a developed 12p - m - lac 
 fractionation platform and evaluated its performance in replicate 
 analysis using reference plasma ( bioreclamation , jericho , ny ) .
first , 
 the loading capacity of the platform was investigated to ensure minimal 
 run - to - run carry over and sample losses , and 25 l of reference 
 plasma volume was determined to be the optimal loading amount .
we 
 then used the optimized loading amount to assess the platform based 
 on total protein recoveries , reproducibility , and efficiency .
the 
 total protein recovery measurements using bca assay ( thermo scientific ) 
 showed an average of 92% of the starting material ( data not shown ) , 
 which agrees with an earlier report .
similarly , 
 coomassie blue stained gels for three analytical replicates of 12p 
 column target ( bound ) proteins and m - lac fractions revealed an identical 
 band pattern and band intensity in replicate samples , indicating good 
 reproducibility of analytical replicates ( supporting 
 information figure 1 ) .
furthermore , we observed gel band differences 
 in the m - lac bound and unbound fractions , suggesting that the m - lac 
 column fractionates the glycoproteome into subpopulations .
12p - depleted plasma fractions were analyzed to evaluate 
 protein abundance changes in proteomics analysis using 1d sds - page 
 and nano - lc 
similarly , glycoproteomics of 12p - m - lac 
 bound and unbound fractions enabled the evaluation of proteins with 
 potential glycosylation changes .
overall , 215 and 248 unique proteins 
 were identified from two analytical replicates in the proteomic and 
 glycoproteomic analyses , respectively .
all proteins reported were 
 identified with 99% confidence ( 1% fdr ) and 2 unique peptides 
 ( supporting information table s2 ) .
details 
 of the distribution of proteins and peptides are presented in supporting information table s3 .
low - abundance 
 glycoproteins and non - glycoproteins identified fell into one of the 
 following functional classification : proteases , lipid associated proteins , 
 cytoskeletal associated proteins , and complement factors ; these functional 
 categories have recently been reported to correlate with disease states . a label - free semiquantitation method based on spectral counts
was 
 utilized to quantify proteins expressed at different amounts in ccrcc 
 plasma samples as previously described . briefly , 1d gel - nano - lc 
ms / ms analysis was performed on equal 
 amounts of 12p - depleted plasma samples followed by data normalization 
 using a reference ratio factor ( total peptide hits of disease / total 
 peptide hits of control ) , as detailed in section 
 2.6 .
in addition , spectral counts were validated 
 by measurements of peak areas of extracted ion chromatograms and manual 
 inspection of ms / ms spectra in random selected cases , as shown in 
 earlier published work .
differentially expressed 
 proteins were selected on the basis of ratios of spectral count fold 
 changes observed between rcc ( + ) and rcc (  ) after normalization . 

potential candidate markers were selected if they were detected in 
 two analytical replicates and exhibited  3 or  3 fold 
 changes , p  0.05 .
as shown in table 1 , the majority ( approximately 74% ) of potential 
 candidate markers were upregulated in the disease proteome .
these 
 include lipid transport and metabolic process proteins ( e.g. , cholesteryl 
 ester transfer protein , apolipoprotein f , apolipoprotein l1 , and phospholipid 
 transfer protein ) , immune system process proteins ( e.g. , basement 
 membrane - specific heparan sulfate proteoglycan core protein , coagulation 
 factor xi , and prostaglandin d2 synthase ( 21kd ) brain ) , and signal 
 transduction proteins ( e.g. , cell surface glycoprotein muc18 , pantetheinase , 
 and junction plakoglobin ) .
we used the gene ontology ( go ) classification 
 system to characterize both the biological process and molecular function 
 of selected potential candidate markers ( figure 2a , b ) .
( a ) molecular function classification , and ( b ) biological 
 process classification . the abundance of both molecular function and 
 biological process is represented by their relative percentage .
average spectral count of two technical 
 replicates . for meaningful ratio calculations , proteins with no spectral 
 counts
 , downregulation ;  , 
 upregulation ; n / a , not identified upon further exploration , 
 we established disease associations and glycosylation status of potential 
 candidate markers using novoseek , a data mining tool resident in the 
 genecards repository ( www.genecards.org ) . the novoseek 
 score , which defines the relevance of the disease to the gene / protein , 
 is based on their literature text - mining algorithms .
disease associations 
 were established on the basis of their scores : the higher the score , 
 the more significant the protein is to the disease .
two unique observations 
 were made : ( 1 ) a strong correlation among potential candidate markers 
 and various kidney and cancer diseases was found and ( 2 ) the majority 
 of potential candidate markers are glycosylated ( table 1 ) .
proteins such as basement membrane - specific heparan sulfate 
 proteoglycan core protein ( hspg2 ) , cell surface glycoprotein muc18 
 ( cd146 ) , l - selectin ( sell ) , vascular cell adhesion protein 1 ( vcam1 ) , 
 and protein z - dependent protease inhibitor ( serpina10 ) have been implicated 
 in several disease states , including clear cell renal cell carcinoma , 
 renal failure , gastric cancer , hepatocellular cancer , prostate cancer , 
 lung cancer , ovarian cancer , breast cancer , and skin cancer .
cd146 , a novel cell adhesion molecule , was recently reported 
 to 
 be a potential marker for clear cell renal cell carcinoma recurrence . 

observed significantly higher levels of cd146 gene expression 
 in patients with metastatic ccrcc compared to that in patients with 
 localized ccrcc and concluded that the recurrence of ccrcc is directly 
 related to the levels of cd146 gene expression . in another publication ,
the presence of cd146 and 
 elevated levels of adiponectin in patients with chronic renal failure 
 were associated with potential indication of endothelial damage and 
 increased cardiovascular risk .
these 
 findings further strengthen our current data wherein a 16-fold abundance 
 increase of cd146 was observed in rcc ( + ) plasma compared to that 
 in rcc (  ) plasma .
future structural studies of cd146 may provide 
 more information in our understanding of the presence of high amounts 
 of cd146 protein and its role in ccrcc plasma .
it is 
 established that changes in m - lac binding affinities ( low or high ) 
 of glycoproteins maybe indicative of the response of glycan structural 
 changes in disease samples .
hence , glycoproteins 
 in m - lac fractions ( bound and unbound ) with glycan alterations were 
 evaluated on the basis of differential m - lac binding .
relative quantification 
 was performed ( see materials and methods ) 
 using spectral counts obtained from ccrcc glycoproteome data . in table 2 , we show a list of glycoproteins with significant 
 differential m - lac binding ( 3 or  3 fold changes , 
 significance level p  0.05 ) and their potential 
 sites of glycosylation based on literature information ( www.uniprot.org ) .
the association between glycan alterations and cancer is well - known , 
 and our current observation of altered glycoproteins is consistent 
 with earlier reports .
average 
 spectral count of two technical 
 replicates . for meaningful ratio calculations , proteins with zero 
 ( 0 ) spectral counts
 , downregulation ; 
  , upregulation ; n / a , not identified for instance , clusterin , a heavily glycosylated protein 
 with seven 
 potential asparagine - linked glycan sites showed increased differential 
 m - lac binding in disease samples vs controls .
clusterin is associated 
 with tumor advancement and carcinogenesis , and recent reports have indicated the presence of clusterin glycan 
 alterations in cancer vs noncancer samples . in stomach cancer studies , 
 bones et al . showed the linearity between decreased levels of clusterin 
 glycans and the progression of cancer .
in addition , clear cell renal cell carcinoma plasma studies revealed 
 significant glycoform changes between rcc ( + ) and rcc (  ) samples 
 of released clusterin glycans .
more recently , 
 we have observed a significant site - specific glycoform alteration 
 of biantennary digalactosylated disialylated ( a2g2s2 ) and core fucosylated 
 biantennary digalactosylated disialylated ( fa2g2s2 ) glycans in disease 
 vs non - diseased ccrcc plasma ( manuscript in preparation ) .
similarly , 
 vitamin d - binding protein ( dbp ) showed increased binding 
 to the m - lac column in ccrcc disease samples .
the relationship between 
 the glycosylation status and function of dbp in cancer patients is 
 still unclear .
earlier data suggested that there is a direct correlation 
 between decreased levels of oligosaccharides present on dbp and inactivity 
 of gc macrophage activating factor ( gcmaf ) in cancer patients .
recently however , rehder et al . investigated dbp glycans levels 
 and observed a high abundance of oligosaccharides in cancer patients , 
 which is in contrast to earlier suggestions .
the current study showed an increase in m - lac binding of dbp in 
 disease compared to non - disease ccrcc samples , suggesting potential 
 glycosylation alterations .
however , the focus of this study was not 
 to investigate the function of dbp ; therefore , further studies are 
 required to provide information on the association of dbp s 
 glycosylation with ccrcc . while gelc 
ms identified interesting 
 proteins based on differential 
 expression , it did not provide information about post - translational 
 modifications ( ptm ) such as glycosylation alterations , and because 
 many biological functions are mediated through glycans , altered glycosylation 
 is a now an established feature of cancer .
therefore , the advantage 
 of m - lac is that it is able to capture some of these subtle changes 
 in order to identify potential biomarkers . in this discovery - based study ,
our goal is to understand 
 the global profile of n - glycans released from low - abundance glycoproteins 
 enriched through depletion of 12 proteins followed by lectin fractionation . 

changes in these low - level glycans may be of potential utility in 
 understanding the presence , progression , and disease recurrence of 
 ccrcc .
to this end , total n - glycans of depleted m - lac fractions of 
 pooled disease rcc ( + ) and non - disease rcc (  ) samples were 
 characterized .
n - glycans were released by pngase f via dot - blotting , 
 online - separated on a porous graphitized carbon ( pgc ) column , and 
 analyzed using lc 
esi tandem mass spectrometry in negative 
 ion mode . utilizing ms retention times , charge states , and ms / ms fragmentation 
 pattern ,
thirty six 
 structurally different n - glycans corresponding to 23 n - glycan monosaccharide 
 compositions were identified from two analytical replicates with minimum 
 variation ( average % cv < 2.5 ) ( figure 3 ) . 

neutral , sialylated ( monosialo , disialo , trisialo , and tetrasialo ) , 
 fucosylated , and high mannose n - glycans were observed .
the identification 
 of n - glycans with various degrees of isomerization was consistent 
 with previous studies of pgc chromatography .
yellow circle , galactose ; 
 blue square , n - acetylglucosamine ; green circle , mannose ; 
 purple diamond , sialic acid ; red triangle , fucose ; core = ( glcnac)2(man)3 .
even though the n - glycan profiles 
 of rcc ( + ) and rcc (  ) 
 m - lac fractions were similar in overall appearance ( supporting information figure s2 ) , a detailed analysis revealed 
 significant differences between disease and non - disease m - lac fractions . 

first , the mean of the relative intensities of the two analytical 
 replicates was calculated , and the data was normalized as previously 
 described .
briefly , the relative intensities 
 of each glycan in a sample was determined using the ratio of the extracted 
 ion chromatography ( eic ) peak area of each n - glycan over the sum of 
 the eic peak areas of all n - glycans in the sample .
total glycan structures 
 were expressed as a percentage after normalization , and relative intensities 
 were established on the basis of relative quantification , not absolute 
 measurements .
the resulting relative abundance of individual n - glycans 
 was compared across different samples .
a detailed list showing the 
 composition , type , isoforms , observed m / z , theoretical mass , and relative intensities ( % ) of n - glycans identified 
 is presented in supporting information table s4 .
following normalization , a comparative qualitative approach 
 was taken to evaluate the 36 observed and normalized n - glycans from 
 rcc ( + ) and rcc (  ) m - lac ( bound and unbound ) fractions .
three 
 unique observations were made ( see supporting 
 information table s4 ) : ( 1 ) sialylated n - glycans were expressed 
 in high levels , and afucosylated disialo - n - glycan , m / z 1111.4 , was observed to be 
 the most abundant glycan structure in all analyzed fractions . in a 
 recent report that involved tissue samples , fucosylated glycans were 
 observed to be the most abundant form , indicating differences in glycan profiles of different disease models . 

afucosylated disialo - n - glycans are a frequently observed occurrence 
 in many cancer glycomic studies . in the present 
 data , we observed two structural isomers for afucosylated disialo - n - glycan 
 with different levels of expression : structure no .
15a with both terminal 
 sialic acid residues in 2,6-linkages eluting prior to structure 
 no .
( 2 ) n - glycan expression levels in m - lac bound fractions were higher 
 compared to that in m - lac unbound fractions , which is consistent with 
 our aim of enriching target glycans using the m - lac column .
in addition , 
 this observation correlates with m - lac s ability to segregate 
 glycan variations into bound and unbound fractions .
( 3 ) the majority 
 of n - glycans were expressed with different amounts when comparing 
 rcc ( + ) and rcc (  ) , and some n - glycan structures ( nos .
13b , 
 18a , 18b , 21b , and 21c ) were observed to be either missing or expressed 
 at extremely low levels in m - lac fractions and therefore were difficult 
 to quantitate .
however , this unique observation may point to a potential 
 glycan - specific molecular feature to differentiate disease and non - disease 
 clear cell renal cell carcinoma plasma samples . after establishing 
 qualitative differences between disease and 
 non - disease fractions , relative quantification and statistical analysis 

were performed to identify differentially expressed n - glycans . for 
 n - glycans with zero ( 0 ) relative abundance
standard student s t - test 
 revealed that 44% of identified n - glycans ( 16 structures ) differ significantly 
 ( average p < 0.011 ) , with an observation of over- 
 or underexpression of n - glycans in rcc ( + ) m - lac fractions .
a notable feature from the differentially expressed glycans was 
 the upregulation of high degree sialylated and high mannose glycans . 

18a , 18b , 21b , 
 and 23b , to be upregulated in disease m - lac fractions ( bound and unbound ) . 

the observation of upregulation of sialylated glycans was recently 
 reported in a study involving tissues of renal cell carcinoma , and our data is in agreement with this report . 

elevated levels of highly branched sialylated glycans are associated 
 with increased activity of sialyltransferases , an enzyme that regulates 
 biosynthesis of sialic acid residues .
several studies 
 have reported an effect of alterations of sialic acids composition 
 on cell adhesion , a factor implicated in metastasis . in breast cancer , for example ,
lin et al . reported that increased 
 amounts of sialic acid correlate with a decrease in cell 
m - lac bound and 
 unbound ; core = 
 ( glcnac)2(man)3 ;  , downregulation ;  , upregulation . 
 for meaningful ratio calculations , glycans with zero ( 0 ) relative 
 abundance
an increased level of high mannose structures ( glycans 
 nos . 1 , 
 9 , and 4 ) was another distinct feature in this study .
elevation of 
 high mannose type glycans have been indicated in various cancer types . 

high levels of high mannose glycans were reported to correlate with 
 breast cancer progression , and a similar 
 trend was reported in head and neck tumor studies .
higher or lower levels of glycans are a hallmark of cancer 
 progression , and their alterations may be related to changes in the 
 expression levels of enzymes involved in the glycan biosynthesis pathway . 
 therefore , the goal of these studies was to assess the potential of 
 n - glycans as biomarker candidates for clear cell renal cell carcinoma .
extracted ion chromatograms 
 ( eics ) of some selected n - glycans were used to validate different 
 amounts of n - glycan expression in m - lac fractions . in figure 4a ,
1 [ core + ( hex)3 ] m / z ( 698.2 ) , n - glycan 
 no . 9 [ core + ( hex)6 ] m / z ( 941.3 ) , n - glycan no . 18a ( isomer a ) [ core + ( hexnac)4(hex)4(neuac)4 ] m / z ( 1178.1 ) , and n - glycan 
 no . 18b ( isomer b ) [ core + ( hexnac)4(hex)4(neuac)4 ] m / z ( 1178.1 ) highlights an elevation 
 of high mannose and tetra - antennary sialo - type oligosaccharides in 
 rcc ( + ) m - lac fractions .
all n - glycan structural identifications were 
 confirmed via ms / ms fragmentation ( supporting 
 information figure s3 ) . in summary ,
our n - glycan data correlates 
 with the glycoproteomics data in which m - lac differential binding 
 suggests potential glycosylation ( glycan - specific ) changes in ccrcc 
 cancer fractions , and our current article marks a novel mapping of 
 glycans in ccrcc plasma .
extracted ion chromatograms to illustrate differentially 
 expressed 
 glycans in m - lac bound and unbound fractions .
9 ( right ) show 
 that both n - glycans are expressed at higher levels in m - lac bound 
 before ccrcc surgery ( upper panels ) compared to those observed after 
 ccrcc surgery ( lower panels ) .
18b ( b ) show that both 
 n - glycans are expressed at higher levels in mlac unbound and bound 
 fractions before ccrcc surgery ( 1st and 3rd panels ) compared to those 
 observed after ccrcc surgery ( 2nd and 4th panels ) .
yellow circle , 
 galactose ; blue square , n - acetylglucosamine ; green 
 circle , mannose ; purple diamond , sialic acid ; red triangle , fucose ; 
 core = ( glcnac)2(man)3 .
we have successfully performed 
 fractionation of pooled plasma from 
 20 patients diagnosed with clear cell renal cell carcinoma ( ccrcc ) 
 using the immuno - affinity depletion of 12 high - abundance proteins 
 and a multi - lectin affinity chromatography ( m - lac ) platform .
alterations 
 in the plasma proteome , glycoproteome , and n - glycome of ccrcc patients 
 were studied for the identification of low - level potential candidate 
 markers that may be of interest for early diagnosis or used as a utility 
 to monitor ccrcc recurrence .
we report that low - abundance proteins 
 with significant expression changes , such as cell surface glycoprotein 
 muc18 , basement membrane - specific heparan sulfate proteoglycan core 
 protein , l - selectin , and vascular cell adhesion protein 1 , may be 
 potential candidates after further validation because of their observed 
 association with various cancers and renal - related diseases .
furthermore , 
 proteins with glycan alterations with differential m - lac column binding 
 confirm reports of the ability of lectins to target potential glyco - biomarker 
 candidates .
complex - type sialylated fucose glycans released from enriched 
 glycoproteins were observed with alterations in ccrcc disease patients 
 that could be related to alterations of glycosylation at the onset 
 of clear cell renal cell carcinoma .
this study was conducted as a 
 first step in identifying potential candidate markers of interest 
 in ccrcc plasma , and the current platform can be well - utilized in 
 the analysis of candidate biomarkers . in the future , we plan to follow 
 up by confirming observed protein abundance and glycan alterations 
 in individual ccrcc patients and validate the observed changes in 
 clinical assays such as elisa , mrm , and antibody  lectin sandwich 
 microarray in a larger cohort .
 clear cell renal cell carcinoma 
 is the most prevalent of all reported kidney cancer cases , and currently 
 there are no markers for early diagnosis . 
 this has stimulated great 
 research interest recently because early detection of the disease 
 can significantly improve the low survival rate . combining the proteome , 
 glycoproteome , and n - glycome data from clear cell renal cell carcinoma 
 plasma 
 has the potential of identifying candidate markers for early 
 diagnosis and prognosis and/or to monitor disease recurrence . here 
 , 
 we report on the utilization of a multi - dimensional fractionation 
 approach ( 12p - m - lac ) and lc  ms / ms to comprehensively investigate 
 clear cell renal cell carcinoma plasma collected before ( disease ) 
 and after ( non - disease ) curative nephrectomy ( n = 
 40 ) . 
 proteins detected in the subproteomes were investigated via label - free 
 quantification . 
 protein abundance analysis revealed a number of low - level 
 proteins with significant differential expression levels in disease 
 samples , including hspg2 , cd146 , ecm1 , sell , syne1 , and vcam1 . 
 importantly , 
 we observed a strong correlation between differentially expressed 
 proteins and clinical status of the patient . 
 investigation of the 
 glycoproteome returned 13 candidate glycoproteins with significant 
 differential m - lac column binding . 
 qualitative analysis indicated 
 that 62% of selected candidate glycoproteins showed higher levels 
 ( upregulation ) in m - lac bound fraction of disease samples . 
 this observation 
 was further confirmed by released n - glycans data in which 53% of identified 
 n - glycans were present at different levels in plasma in the disease 
 vs non - disease samples . 
 this striking result demonstrates the potential 
 for significant protein glycosylation alterations in clear cell renal 
 cell carcinoma cancer plasma . with future validation in a larger cohort , 
 
 information derived from this study may lead to the development of 
 clear cell renal cell carcinoma candidate biomarkers . 
Introduction
Materials and Methods
Results
and Discussion
Conclusions