the 
 discovery of biomarkers useful for the early detection of epithelial 
 ovarian cancer ( eoc ) has been complicated by several factors : ( i ) 
 there are different histological subtypes of eoc that have very unique 
 genetic alterations , ( ii ) the tissues and serum samples available 
 for biomarker discovery are frequently collected from patients in 
 later stages of the disease , and ( iii ) the expected sensitivity and 
 specificity required for a biomarker to detect eoc is high due to 
 the lower disease incidence and high risk surgical interventions . 
 despite these obstacles ,
proteomic studies profiling potential biomarkers 
 for eoc have the potential to increase our knowledge about this disease , 
 leading to improvements in clinical disease management .
glycoproteins 
 account for 80% of the proteins located at 
 the cell surface and in the extracellular environment .
therefore , 
 strategies to identify glycoproteins that have tumor - specific glycosylation 
 changes could lead to the discovery of novel diagnostic and therapeutic 
 markers . in our present study
, we are targeting the neutral n - linked 
 glycan structure known as bisecting glycosylation that is amplified 
 in ovarian cancer tissues , focusing on membrane proteins .
membrane 
 proteins play pivotal roles in regulating signaling pathways , and 
 these proteins can frequently be found both on the cell surface and 
 in circulation due to enzyme release making them ideal potential biomarkers .
bisecting glycosylation , added by the glycosyltransferase known 
 as gnt - iii , is normally expressed on glycoproteins in the brain and 
 kidney .
the functional effects of amplified 
 bisecting glycosylation on the growth and spread of ovarian cancer 
 have not been determined previously . in this manuscript , we have identified 
 membrane proteins with bisecting glycosylation from human primary 
 endometrioid and serous ovarian cancer tissues and determined the 
 glycan structures from these proteins using mass spectrometry methods . 

functional annotation of this data demonstrates that membrane proteins 
 with bisecting glycosylation in ovarian cancer have significant functions 
 including : cell adhesion , control of immune responses , regulation 
 of protein transport , and participation in signaling pathways such 
 as notch , wnt , and tgf. also , our glycan structural analysis 
 from primary ovarian tumor tissues demonstrates that gnt - iii overexpression 
 in human ovarian cancer produces bisected n - linked glycans that have 
 dramatic reductions in branching and extensions and may be useful 
 as a biomarker for the development of therapeutic and diagnostic advances .
biotinylated e - pha ( phaseolus vulgaris erythroagglutinin ) and streptavidin - hrp were obtained from vector 
 laboratories ( burlingame , ca ) .
the egfr receptor antibody , ha antibody , 4g10 antibody , and 
 all secondary antibodies were obtained from santa cruz biotechnologies . 

the streptavidin - fitc , egf - fitc , and eea1 antibody were obtained from 
 invitrogen .
the mouse ha - tagged gnt - iii expression construct was a 
 gift from dr .
fresh frozen human endometrioid ovarian cancer 
 ( eoc ) tissues ( n = 3 ) , human serous ovarian cancer 
 tissues ( n = 3 ) , normal human ovary tissues ( n = 2 ) , and isolated ovarian surface epithelial cells ( 100 
 mg each ) were obtained from the ovarian cancer institute ( atlanta , 
 ga ) .
institutional review board approval for this research was obtained 
 from the georgia institute of technology , the university of georgia , 
 northside hospital ( atlanta , ga ) and the university of arkansas for 
 medical sciences .
tissue or cells ( 100 mg ) were homogenized 
 in 10 mm hepes ph 7.5 . 

the slurry was placed in a dounce glass homogenizer , and cells were 
 lysed with 10 strokes of the large and fine pestle .
the solution was 
 incubated on ice for 1 h to continue the hypotonic cell lysis .
the supernatant was placed in a beckman ultracentrifuge tube , and 
 the solution was centrifuged at 100  000 g for 
 1 h at 4 c to isolate a total membrane pellet .
the pellet was 
 rinsed and sonicated into 40 mm ammonium bicarbonate/10 mm dtt at 
 room temperature for 2 h to reduce proteins .
an equal volume of iodoacetamide 
 ( 10 mg / ml ) in 40 mm ammonium bicarbonate was added , and the solution 
 was mixed by vortexing and placed in the dark for 45 min .
the solution 
 was dialyzed into 10 mm ammonium bicarbonate using tube - o  dialyzer 
 membranes overnight at 4 c .
protein concentration was measured 
 using bca assay , and 500 g of total proteins was used for e - pha 
 capture , while 60 g was digested with sequencing - grade trypsin 
 overnight for prelectin nano - esi  rplc  ms / ms analysis . 

sodium chloride was added to each e - pha capture sample ( final 150 
 mm ) , 5 mm calcium chloride , 5 mm magnesium chloride , and 10 g 
 biotinylated e - pha lectin prior to overnight incubation at 4 c . 

lectin - bound proteins were captured using 100 l of paramagnetic 
 streptavidin particles ( promega ) at 4 c for 2 h. beads were 
 washed three times with 10 mm ammonium bicarbonate supplemented with 
 salts .
proteins were eluted from beads using 4 m urea/4 mm dtt/40 
 mm ammonium bicarbonate at 50 c for 1 h. proteins were digested 
 with 5 g of sequencing - grade trypsin overnight at 37 c . 

peptides were acidified to a final of 0.1% trifluoroacetic acid and 
 purified using c18 spin columns .
peptides were dried in a speedvac 
 prior to resuspension in 19.5 l of buffer a ( 0.1% formic acid ) 
 and 0.5 l of buffer b ( 75% acetonitrile , 0.1% formic acid ) . 

ms / ms with a 
 thermo orbitrap velos mass spectrometer equipped with a waters nanoacquity 
 lc system .
tryptic peptides were separated by reverse - phase jupiter 
 proteo resin ( phenomenex ) on a 100  0.1 mm column using a nanoacquity 
 uplc system ( waters ) .
peptides were eluted using a 120 min gradient 
 from 98:2 to 40:60 buffer a / b ratio .
buffer a = 0.1% formic acid , 0.05% acetonitrile , 
 buffer b = 0.1% formic acid , 75% acetonitrile .
eluted peptides were 
 ionized by electrospray ( 2.0 kv ) , followed by ms / ms analysis using 
 collision - induced dissociation on a thermo ltq orbitrap velos mass 
 spectrometer .
ms data were acquired using the ftms analyzer in a profile 
 mode at a resolution of 60  000 over a mass range of 375 to 
 1500 m / z .
ms / ms data were collected 
 from the top 15 peaks from each ms scan using the ion trap analyzer 
 in centroid mode and normal mass range with normalized collision energy 
 of 35.0 with a dynamic exclusion of two repeat counts at 30 s duration . to facilitate the 
 analysis of oligosaccharides by ms , n - linked glycans from e - pha captured 
 glycoproteins were released by n - glycanase and permethylated as described 
 previously .
the resulting digests 
 were enriched for glycopeptides prior to pngasef ( prozyme ) treatment 
 to release the n - linked glycans .
contaminating peptides and salts 
 were removed using sep - pak c18 chromatography , and the resulting glycans 
 were permethylated prior to analysis using a linear ion trap mass 
 spectrometer ( ltq orbitrap ; thermo scientific ) .
detection and relative 
 quantification of bisecting n - linked glycan structures were carried 
 out by using the total ion mapping ( tim ) function of the xcalibur 
 software package version 2.0 ( thermo fisher scientific ) as described 
 previously .
the tim profiles were initially 
 filtered with neutral loss of terminal hexnac (  mass , 260 ( 1 + ) , 
 130 ( 2 + ) , and 86.7 ( 3 + ) ) to assess the presence of bisecting n - linked 
 glycan structures .
n - glycan structures carrying terminal hexnac were 
 further subjected to manual msn analysis to determine bisecting n - linked 
 glycan structures .
raw peptide 
 data were converted 
 to mzxml , and ms / ms spectra were searched against the international 
 protein index ( ipi ) human database ( ipi.human.v.3.71 ) using myrimatch .
idpicker incorporates searches against a separate reverse database , 
 probability match from myrimatch , and deltcn scores to achieve a false - discovery 
 rate of < 5% .
variance 
 for membrane protein extractions and e - pha capture between normal 
 and tumor tissues were calculated by measuring the number of peptides 
 identified for proteins that adhere to e - pha from membrane extractions 
 in a non - glycan - dependent manner such as hspa2 and rpl9 .
values observed 
 for tumor tissues for hspa2 ( cv 0.63/8 peptides ) and rpl9 ( cv 1.36/4 
 peptides ) .
values observed for normal tissues hspa2 ( cv 0.71/7 peptides ) 
 and rpl9 ( cv 1.4/4 peptides ) .
these results indicate a 12  1.0% 
 variance between normal and tumor samples due to processing .
this 
 variance is well below the 1.5 times or 150% increase in peptides 
 and spectral counts we have established as criteria to be considered 
 as a glycoprotein with tumor - specific e - pha reactivity .
proteins in table 1 were uploaded to david 2009 ( the database for annotation , 
 visualization and integrated discovery ) for analysis .
tumor - specific e - pha enrichment 
 required 1.5 times increase in spectral counts and peptide abundance 
 in tumor tissues compared with nonmalignant tissues .
one sequence gave > 75% 
 reduction 
 in gnt - iii expression in human ovarian adenocarcinoma cells ( nm_002409 , 
 5 ctacgaccggttccactac ) .
briefly , 
 the target sequence along with restriction site linkers and shrna 
 loop sequence was cloned into the psih - h1shrna vector ( system biosciences ) . 

lentivirus was produced in 293tn cells , and ovcar3 cells were transduced 
 as previously described .
ovcar3 cells 
 stably expressing gnt - iii sirna target sequence were transfected transiently 
 with ha - tagged mouse gnt - iii cdna expression plasmid ( gift from pamela 
 stanley ) or empty cdna vector .
subconfluent cells were serum - starved 
 for 24 h prior to the addition of 25 ng / ml egf for 24 h. cell proliferation 
 was measured using the celltiter aqueous one solution ( promega ) according 
 to the manufacturer s recommendations and absorbance was read 
 at 490 nm .
cells were 
 grown to subconfluence 
 in chamber slides before fixing in 3% formaldehyde/1 phosphate 
 buffered saline ( pbs ) for 10 min .
cells were blocked using 1% bovine 
 serum albumin ( bsa)/1xpbs-0.2% tween-20 ( pbst ) prior to the addition 
 of biotin - labeled e - pha ( vector laboratories ) diluted 1:300 in 1%bsa/1xpbst . 

bound lectin was detected using fluorescein - tagged streptavidin ( invitrogen ) 
 diluted 1:500 in 1% bsa/1xpbst .
cells were placed on ice for 
 1 h in the presence of fluorescein tagged egf ( 50 ng / ml ) ( invitrogen ) . 

cells were placed back at 37 c for 30 min before the removal 
 of unbound egf with ice - cold 20 mm acetic acid/0.5 m nacl .
cells were 
 fixed prior to detection of early endosomes using anti - eea1 ( 1:400 ) 
 ( invitrogen ) , followed by rhodamine antimouse igg ( 1:250 ) .
in 
 previous studies we have used the plant lectin e - pha ( phaseolus 
 vulgaris erythroagglutinin ) to capture soluble or extracellular 
 glycoproteins with bisecting glycosylation from human ovarian cancer 
 tissues due to the elevation of this form of glycosylation in human 
 epithelial eoc . in this study
, we are comparing 
 the analysis of both serous and endometrioid ovarian cancer , the two 
 most abundant histological subtypes of eoc , focusing on the identification 
 of glycoproteins located in the membrane fraction that have tumor - specific 
 bisecting glycosylation .
bisecting glycosylation is amplified in ovarian 
 cancer due to increased expression of the glycosyltransferase gnt - iii .
therefore , to explore the localization of glycoproteins with bisecting 
 glycosylation and validate the specificity of e - pha for this glycan 
 structure , we have created cell lines using the human ovarian adenocarcinoma 
 cell line known as ovcar3 that express either a control shrna sequence 
 that targets no human gene ( control sirna ) or a gnt - iii shrna target 
 sequence ( gnt - iii sirna ) .
constitutive expression of the gnt - iii sirna 
 reduced the expression of gnt - iii by > 75% compared with control 
 sirna 
 ( figure 1a ) .
e - pha binding in ovcar3 cells 
 stably expressing the control sirna is strong , localized to the cell 
cell 
 junctions , indicating a concentration of glycoproteins at the cell 
 periphery with bisecting glycosylation .
the cell boundaries of ovcar3 
 cells stably expressing gnt - iii sirna are not visible , indicating 
 a loss of e - pha accumulation and a reduction of bisecting glycosylation 
 ( figure 1b ) . therefore , e - pha binding to glycoproteins 
 in ovcar3 cells requires the expression of gnt - iii , and glycoproteins 
 with bisecting glycosylation are concentrated at the cell periphery .
e - pha 
 binds bisecting glycans in a gnt - iii dependent manner . ( a ) 
 real - time pcr measurement of gnt - iii mrna levels in ovcar3 human ovarian 
 cancer cells expressing control sirna or gnt - iii sirna .
gnt - iii levels 
 were normalized to rpl4 and the control sirna level was set to 1.0 
 for comparison ; error bars represent the mean  se for triplicate 
 measurements .
( b ) e - pha staining of glycoproteins with bisecting glycosylation 
 in ovcar3 cells ; nuclei are stained with dapi , magnification 20. fresh frozen tissue 
 samples ( supplementary table 1 in the supporting 
 information ) from eoc ( n = 3 ) , serous ovarian 
 cancer ( n = 3 ) , nonmalignant ovary ( n = 2 ) , or isolated nonmalignant ovarian surface epithelial cells 
 ( n = 1 ) were each subjected to membrane protein isolation 
 and e - pha lectin capture .
a diagram of the extraction and analysis 
 scheme for glycoproteomics is shown in figure 2a .
comparison analysis of each membrane extracted sample before and 
 after e - pha capture enabled the identification of glycoproteins that 
 are potential acceptors for bisecting glycosylation .
glycoproteins 
 that were enriched following e - pha capture by at least 1.5 times ( 150% 
 by spectral counts ) in tumor tissues relative to nonmalignant controls 
 are listed in table 1 . in table 1
, we provide human protein atlas ( hpa ) antibody immunohistochemistry staining data ( for ovarian 
 cancer tissues ) for each e - pha enriched glycoprotein . over 80% of 
 the glycoproteins in table 1 have positive 
 antibody immunohistochemistry staining in ovarian carcinoma tissues 
 ( data from the hpa annotated in table 1 ) . the majority ( 81% ) of glycoproteins enriched 
 by e
- pha are present in both histological subtypes of eoc , suggesting 
 that the glycosylation changes on these proteins are important for 
 the survival and growth of ovarian cancer cells ( figure 2b ) .
the overall functional annotation ( figure 2c ) of glycoproteins enriched by e - pha capture reveals that 
 the largest functional categories are cell adhesion , regulation of 
 signaling , enzymatic functions , and control of protein transport ( figure 2c ) .
several glycoproteins have reported activities 
 in the notch ( lrp2 , postn , thbs1 , thbs2 , mfap4 , olfm4 , ncstn ) , tgf,(aspn , fbn1 , eng ) , and wnt ( col1a1 , 
 ctnnb1 ) signaling pathways .
membrane proteins 
 were analyzed by nanoflow - esi  rplc  ms / ms before e - pha 
 capture to verify equivalent protein quality and quantity .
biotinylated 
 e - pha was added to membrane extracted proteins , and glycoproteins 
 with bisecting glycans were captured using streptavidin magnetic resin 
 prior to nanoflow - esi  rplc  ms / ms .
membrane glycoproteins 
 enriched in tumor tissues relative to nonmalignant tissues by e - pha 
 capture by at least 1.5 times or 150% by spectral counts were identified 
 and are listed in table 1 .
( b ) venn diagram 
 showing the percentage of glycoproteins in table 1 from endometrioid only ( 13% purple ) , serous only ( 6% blue ) , 
 or shared ( 81% blue / purple ) .
( c ) functional annotation of e - pha enriched 
 membrane glycoproteins using david 2009 .
the membrane - extracted proteins 
 from endometrioid and serous ovarian tissues were subjected to pngasef 
 treatment following e - pha capture .
membrane proteins extracted from 
 nonmalignant ovarian surface epithelial cells have low levels of binding 
 to e - pha ; therefore , equivalent levels of total membrane protein were 
 used for the release of n - linked glycans .
the n - linked glycans were 
 released from trypsin and chymotrypsin digests using pngasef before 
 permethylation and analysis using mass spectrometry methods .
we have 
 identified nine very unique and unusual bisecting n - linked glycan 
 structures that are present in both endometrioid and serous eoc tissues 
 ( figures 3 and 4 ) that 
 were not observed in control nonmalignant ovarian epithelial cells . 

the full ms reveals the presence of abundant high mannose glycan structures 
 ( supplemental figure 1 in the supporting information ) that are marked with asterisks in figure 3 .
ms6 analysis was necessary to confirm the presence of the bisecting 
 glycan structures ( supplemental figures 24 
 in the supporting information ) .
the fragment ion observed at m / z 444 for ms6 clearly demonstrates the 
 presence of the bisected mannose , of which three of the hydroxyl groups 
 were substituted with three hexnac ( supplemental 
 figures 24 in the supporting information ) .
although , 
 some of the bisecting n - linked glycan structures contain isobaric 
 forms of n - glycans capped with terminal hexnac , which demonstrates 
 a specific fragment ion at m / z 458 
 for ms6 ( supplemental figure 2 in the supporting 
 information ) .
thus , the sequential msn approach using permethylated 
 n - glycans effectively discriminates bisecting n - glycan structures 
 from other n - glycans with terminal hexnac .
the most prevalent bisecting 
 n - linked structures are shown on the tim chromatogram ( figure 3 ) .
these structures are dramatically truncated with 
 minimal branching with reductions in distal galactose and sialic acid 
 modifications .
most of the bisecting n - linked glycans also have core 
 fucosylation , another neutral n - linked glycan structure known to be 
 amplified in eoc .
the unusual lack of branched 
 glycans with the presence of core fucosylation may represent a biomarker 
 useful for the development of future therapeutic and diagnostic applications .
prevalence 
 of unique bisecting n - linked glycan structures in ovarian 
 carcinoma tissues .
pngasef released n - linked glycan structures from 
 glycoproteins enriched by e - pha capture . glycans were released from 
 pooled e - pha captured glycoproteins from endometrioid ( n = 3 ) , serous ( n = 3 ) , or total membrane proteins 
 from nonmalignant control surface epithelial cells ( n = 1 ) .
tim profile was filtered with neutral loss of hexnac and bisecting 
 n - linked glycan structures were identified by msn analysis manually . 

peaks labeled m7 , m8 , and m9 indicate high mannose type n - linked glycans . bisecting n - linked glycan structures identified 
 from human endometrioid 
 and
bisecting structures identified from endometrioid 
 and serous ovarian cancer tissues by msn analysis are summarized
. 
 represented glycan structures are in accordance with the guidelines 
 from the consortium for functional glycomics ( cfg ) : blue square , n - acetylglucosamine ( glcnac ) ; green circle , mannose ( man ) ; 
 yellow circle , galactose ( gal ) ; red triangle , fucose ( fuc ) .
our glycoproteomic analysis 
 of e - pha captured membrane glycoproteins 
 from eoc has led to the identification of potential functions that 
 bisecting glycosylation changes may impact .
several proteins enriched 
 by e - pha have potential functions in the control of protein transport 
 ( bcap31 , lman1 , lman2 , lrp2 , myof , sec22b , ssr4 , and tmed10 ) . to explore 
 this area of functional significance
further , we exposed the ovcar3 
 cell lines , control sirna , or gnt - iii sirna to fluorescently tagged 
 egf to track the transport of receptors that receive bisecting glycosylation 
 and bind egf , such as egfr .
egf was internalized in both cells ; however , 
 in control cells , the egf is distributed to several intracellular 
 vesicles , and in gnt - iii sirna cells , the egf is primarily localized 
 in a perinuclear compartment ( figure 5 ) .
egf 
 is active in the cells demonstrated by the ligand - induced phosphorylation 
 observed ( supplementary data in the supporting 
 information , figure 5 , last panel ) . 

the early endosomal marker eea1 also displays a change in intracellular 
 localization in gntiii sirna cells compared with control cells .
these 
 results suggest that bisecting glycosylation is promoting transport 
 of certain proteins in ovarian cancer cells . to confirm that this 
 protein transport change in gnt - iii sirna cells is due to bisecting 
 glycosylation
, we show that we can rescue this defect with the expression 
 of mouse gnt - iii in gnt - iii sirna stable ovcar3 cells .
mouse gnt - iii 
 is not targeted by our constitutively expressed human gnt - iii shrna 
 target sequence .
stable gnt - iii sirna ovcar3 cells were transiently 
 transfected with full - length mouse gnt - iii fused to an ha - tag .
immunofluorescence 
 microscopy demonstrates that cells transiently transfected show positive 
 e - pha staining ( green , fitc , figure 6a ) ) and 
 positive ha - tag staining ( red , rhodamine , figure 6a ) , indicating that the mouse gnt - iii is active in human cells . 

introduction of the mouse gnt - iii gene into ovcar3 cells stably expressing 
 gnt - iii sirna can rescue the transport of egf - bound glycoproteins 
 ( figure 6b ) .
fluorescent egf and rhodamine - stained 
 eea1 are transported into different intracellular vesicles in rescued 
 cells ( figure 6b top panel ) , while egf and 
 eea1 show stalled movement in a perinuclear compartment without rescue 
 in the gnt - iii sirna cells without mouse gnt - iii ( figure 6b , bottom panel ) .
these experiments confirm a novel 
 role for gnt - iii expression and therefore bisecting glycans in promoting 
 vesicular transport in human ovarian adenocarcinoma cells .
downstream 
 signaling induced by egf is suppressed in gnt - iii sirna cells ( supplementary data in the supporting information , figure 6 ) .
ovcar3 cells stably expressing 
 gnt - iii sirna have reduced basal proliferation and show no change 
 in cell proliferation following egf addition ( figure 6c ) .
transfection of mouse gnt - iii increases basal cell proliferation 
 and restores the proliferation response to egf ( figure 6c ) .
these data indicate that gnt - iii expression and bisecting 
 glycosylation promote the vesicular transport and activity of egf - bound 
 receptors such as egfr .
( a ) ovcar3 
 control sirna cells and gnt - iii sirna cells were serum - starved prior 
 to incubation with fluorescein - egf ( 50 ng / ml ) for 1 h on ice .
cells 
 were placed in a 37 c incubator for 30 min prior to removal 
 of egf that was not internalized by an acid wash .
nuclei are stained 
 with dapi , magnification 40. protein transport and response to egf is restored with mouse gnt - iii 
 expression .
( a ) ovcar3 cells expressing gnt - iii sirna constitutively 
 were transiently transfected with mouse ha - gnt - iii ( gift from pamela 
 stanley ) .
cells expressing mouse ha - gnt - iii show positive e - pha staining 
 and ha antibody staining indicating expression and activity in human 
 cells .
nuclei are stained with dapi , magnification 20. ( b ) transport 
 of egf fluorescein - bound receptors is rescued by mouse ha - gnt - iii 
 expression .
ovcar3 gnt - iii sirna cells were transiently transfected 
 with mouse ha - gnt - iii ( top panel ) or empty vector ( bottom panel ) . 

cells were serum - starved for 2 h 1 day after transfection prior to 
 incubation with fluorescein - egf , as described for figure 4 .
( c ) ovcar3 gnt - iii sirna cells were transiently 
 transfected with vector - only or mouse ha - gnt - iii prior to plating 
 into 96-well plates .
cells were serum - starved and incubated with vehicle 
 only or egf 35 ng / ml for 24 h. cell proliferation was measured using 
 celltiter aqueous one ( promega ) reagent .
the mechanisms that lead to overexpression 
 of gnt - iii in ovarian 
 cancer may involve epigenetic mechanisms .
a recent study of human ovarian cancer and nonmalignant ovarian 
 epithelial cell lines demonstrated that the mgat3 gene could be induced following treatment with an inhibitor of dna 
 methyltransferase .
these results provide strong evidence that the 
 increased expression of mgat3 transcripts in ovarian 
 cancer may be due to epigenetic dysregulation of hypermethylation . the normal functional roles of bisecting glycans on glycoproteins 
 in the kidney and brain are not well understood .
the overexpression 
 of gnt - iii in ovarian cancer offers an opportunity to learn more about 
 this form of glycosylation and discover new mechanisms that contribute 
 to ovarian tumor growth .
we have discovered a new function for 
 gnt - iii expression and bisecting 
 n - linked glycosylation in promoting protein transport .
suppression 
 of gnt - iii expression in the human ovarian cancer cell line ovcar3 
 results in a dramatic change in the intracellular localization of 
 egf bound receptors .
we have confirmed that egfr is glycosylated by 
 gnt - iii in ovcar3 cells ( supplementary data in 
 the supporting information , figure 5 , top panel ) .
egf was capable of activating the egfr receptor , and 
 there are no differences in egfr autophosphorylation levels between 
 control sirna and gnt - iii sirna ovcar3 cells ( supplementary figure 5 in the supporting information ) , suggesting 
 that receptor dimerization and endocytosis are not influenced by bisecting 
 glycosylation .
however , the transport of vesicles containing egf bound 
 receptor is slowed in cells with reduced gnt - iii expression , allowing 
 the egf to accumulate in a perinuclear compartment .
the accumulation 
 of egf - bound receptor in this perinuclear compartment reduces downstream 
 egf - induced signaling ( supplementary data in the 
 supporting information , figure 6 ) and 
 inhibits proliferative responses to egf ( figure 6c ) . our ability to rescue the transport and activity of egf - bound 
 receptors with the expression of mouse ha - gnt - iii ( figure
6a  c ) supports a role for gnt - iii expression 
 and bisecting glycosylation as a factor promoting growth factor receptor 
 transport and activity .
our glycoproteomic analysis from ovarian cancer 
 tissues also supports a role for bisecting glycans in protein transport 
 as several proteins with known transport functions are enriched by 
 e - pha .
therapeutic strategies to block gnt - iii expression , enzymatic 
 activity , or growth factor receptor interactions with transport proteins 
 may be new avenues to reduce ovarian cancer cell responses to growth 
 factor ligands by slowing the movement of receptors within the cell .
the unusual bisecting n - linked glycan structures identified for 
 both endometrioid and serous eoc subtypes are an important discovery 
 that may lead to the development of antibodies or strategies to target 
 ovarian cancer cells .
the prevalence and similarity of these glycan 
 structures for both histological subtypes of ovarian cancer indicate 
 an importance for this glycan structure in the development and survival 
 of ovarian carcinoma cells .
also , the lack of these structures in 
 nonmalignant tissues and cells supports the use of these structures 
 as potential biomarkers .
a recent glycomic analysis of all n - linked 
 glycan structures for membrane glycoproteins isolated from ovarian 
 cancer and nonmalignant ovarian cell lines also found the presence 
 of bisecting structures specific to malignant cells .
gnt - iii expression and bisecting glycosylation have been 
 reported to compete with branching enzymes such as gnt - v , reducing 
 tumor growth for cancers with elevated gnt - v expression such as breast 
 cancer .
however , in ovarian cancer cells 
 there is no competition with gnt - v , and the bisecting glycan structure 
 has a growth promoting role .
furthermore , the truncated glycan structures 
 found on bisecting n - linked glycans released from ovarian cancer tissues 
 have not been observed in analysis of bisecting n - linked glycan structures 
 isolated from nonmalignant cell types such as hek293 and cho .
these results indicate that there may be additional changes in golgi 
 transport or other as yet unknown factors in ovarian cancer that may 
 contribute to the production of these unusual glycan structures .
several of the extracellular glycoproteins identified following 
 the stringent membrane isolation procedures ( thbs1 , thbs2 , lrp2 , postn ) 
 are adhesive glycoproteins with reported functions as noncanonical 
 regulators of the notch signaling pathway .
the extraction of these proteins with membrane protein fractions 
 suggests high affinity interactions with membrane proteins .
we have 
 identified nicastrin ( ncstn ) as a glycoprotein enriched by e - pha specific 
 to serous ovarian cancer .
the notch signaling pathway has been identified by the cancer genome 
 atlas ( tcga ) as a pathway significant for the pathophysiology of ovarian 
 cancer .
the abundance of glycoproteins 
 known to participate in the notch signaling pathway that are enriched 
 by e - pha capture in tumor tissues suggest that this unusual bisecting 
 glycan structure may be important for notch functions in ovarian carcinoma .
high - grade serous ovarian cancer is the largest fraction of ovarian 
 cancer patients ( 80% ) .
the small percentage of membrane glycoproteins 
 that we identified that are unique to this group of cancers following 
 e - pha capture may offer some insights into why these tumors are so 
 aggressive .
the bst2 ( cd317 ) is a tetherin glycoprotein that 
 is linked to the membrane via both transmembrane and glycosylphosphatidylinositol 
 ( gpi ) linkage .
this protein is normally expressed in very specialized 
 cells such as paneth cells and in the bone marrow with functions as 
 an intrinsic immunity factor .
the use 
 of this protein as a therapeutic biomarker for multiple myeloma has 
 been complicated due to expression in nonmalignant bone marrow cells .
our discovery of a unique form of glycosylation 
 on this protein in serous ovarian cancer is novel and may allow use 
 of bst2 as a diagnostic or therapeutic target for this solid tumor .
endoglin or cd105 is another glycoprotein unique 
 to the serous ovarian carcinoma tumor tissues .
a recent study identified endoglin as a potential gpi anchored protein 
 biomarker for breast cancer .
the gpi linkage 
 may explain the increased levels of soluble endoglin in the ascites 
 of ovarian cancer patients .
therefore , the increased expression levels of endoglin in ovarian 
 cancer coupled to the presence of unique bisecting glycosylation make 
 endoglin a potential therapeutic target for serous ovarian carcinoma .
cadherin-13 or t ( truncated ) cadherin is an atypical 
 gpi - linked cadherin .
cdh13 has been referred to as a tumor suppressor 
 that is dysregulated by epigenetic changes in methylation for many 
 cancers including ovarian cancer . however
, 
 cdh13 has also been demonstrated as a stromal factor that is expressed 
 in the tumor microvascular that can favor tumor growth .
also , cdh13 has been shown to associate with 
 cell surface integrins and grp78 to promote cell survival signaling 
 pathways .
our identification of cdh13 
 with serous ovarian carcinoma membranes indicates that this protein 
 is expressed in serous ovarian cancer .
future studies will be needed 
 to study the functional impact of cdh13 in ovarian cancer cell survival .
nicastrin is a component of the -secretase 
 ( gs ) multiprotein enzyme complex responsible for activation of various 
 cell surface membrane proteins including notch .
nicastrin has been 
 shown to participate in the regulation of breast cancer stem cell 
 expansion and tumor growth .
our identification 
 of nicastrin as a glycoprotein with bisecting glycosylation in serous 
 ovarian carcinoma is novel , and future studies will be needed to determine 
 the role of nicastrin and bisecting glycosylation on ovarian cancer 
 growth and survival . in conclusion
, we present evidence that 
 gnt - iii expression is required for the expression of bisecting n - linked 
 glycosylation detected using the lectin e - pha .
because of the elevated 
 expression levels of gnt - iii in ovarian cancer , we have utilized e - pha 
 for the glycoproteomic analysis of membrane proteins extracted from 
 primary ovarian carcinoma tissues ( serous and endometrioid ) .
we identified 
 100 proteins that were enriched in abundance in tumors demonstrated 
 by an increase in spectral counts by at least 1.5 times following 
 e - pha capture .
our results suggest new functional roles for bisecting 
 glycosylation in controlling the growth and survival of ovarian cancer . 

these results may lead to future studies that contribute to the development 
 of new therapeutic interventions for the treatment of ovarian cancer .
 biomarkers capable of detecting and 
 targeting epithelial ovarian 
 cancer cells for diagnostics and therapeutics would be extremely valuable . 
 
 ovarian cancer is the deadliest reproductive malignancy among women 
 in the u.s . 
 , killing over 14  000 women each year . 
 both the 
 lack of presenting symptoms and high mortality rates illustrate the 
 need for earlier diagnosis and improved treatment of this disease . 
 
 the glycosyltransferase enzyme gnt - iii encoded by the mgat3 gene is responsible for the addition of glcnac ( n - acetylglucosamine ) to form bisecting n - linked glycan structures . 
 
 gnt - iii mrna expression is amplified in ovarian cancer tissues compared 
 with normal ovarian tissue . 
 we use a lectin capture strategy coupled 
 to nano - esi  rplc  ms / ms to isolate and identify the membrane 
 glycoproteins and unique glycan structures associated with gnt - iii 
 amplification in human ovarian cancer tissues . 
 our data illustrate 
 that the majority of membrane glycoproteins with bisecting glycosylation 
 are common to both serous and endometrioid histological subtypes of 
 ovarian cancer , and several have been reported to participate in signaling 
 pathways such as notch , wnt , and tgf. 
Introduction
Materials
and Methods
Results
Discussion