Glycoproteomic Analysis of the Secretome ofHuman Endothelial Cells*□S
Xiaoke Yin‡, Marshall Bern§, Qiuru Xing‡, Jenny Ho¶, Rosa Viner�, and Manuel Mayr‡**
Previous proteomics studies have partially unraveled thecomplexity of endothelial protein secretion but have notinvestigated glycosylation, a key modification of secretedand membrane proteins for cell communication. In thisstudy, human umbilical vein endothelial cells were kept inserum-free medium before activation by phorbol-12-my-ristate-13 acetate, a commonly used secretagogue thatinduces exocytosis of endothelial vesicles. In addition to123 secreted proteins, the secretome was particularly richin membrane proteins. Glycopeptides were enriched byzwitterionic hydrophilic interaction liquid chromatographyresins and were either treated with PNGase F and H2
18Oor directly analyzed using a recently developed workflowcombining higher-energy C-trap dissociation (HCD) withelectron-transfer dissociation (ETD) for a hybrid linear iontrap–orbitrap mass spectrometer. After deglycosylationwith PNGase F in the presence of H2
18O, 123 unique pep-tides displayed 18O-deamidation of asparagine, corre-sponding to 86 proteins with a total of 121 glycosylationsites. Direct glycopeptide analysis via HCD-ETD identified131 glycopeptides from 59 proteins and 118 glycosylationsites, of which 41 were known, 51 were predicted, and 26were novel. Two methods were compared: alternatingHCD-ETD and HCD-product-dependent ETD. The formerdetected predominantly high-intensity, multiply chargedglycopeptides, whereas the latter preferentially selectedprecursors with complex/hybrid glycans for fragmenta-tion. Validation was performed by means of glycoproteinenrichment and analysis of the input, the flow-through,and the bound fraction. This study represents the mostcomprehensive characterization of endothelial protein se-cretion to date and demonstrates the potential of newHCD-ETD workflows for determining the glycosylationstatus of complex biological samples. Molecular & Cel-lular Proteomics 12: 10.1074/mcp.M112.024018, 1–23,2013.
Cardiovascular disease manifests predominantly as myo-cardial ischemia, heart failure, stroke, aortic aneurysm, and
peripheral vascular disease and leads to the majority ofdeaths and disabilities worldwide. Endothelial cells (ECs) con-stitute the inner lining of all blood vessels and form the inter-face between the circulation and the vascular wall (1). Theendothelial monolayer is pivotal for maintaining vascular ho-meostasis through a balance of endothelium-derived factors(2, 3). ECs are preferred targets of cardiovascular risk factorssuch as hypercholesterolemia, diabetes, hypertension, andsmoking (1, 4). Repetitive injury is associated with a varyingdegree of endothelial dysfunction. Alterations in its anticoag-ulant and anti-inflammatory properties leave the vasculaturesusceptible to disease (5) and play a key role in the initiationand progression of cardiovascular disease (6).
Previous proteomics studies (7–13), including one by ourgroup (8), have investigated the secretome of unstimulatedhuman umbilical vein ECs (HUVECs), the most widely usedECs in cardiovascular research. Only two studies have ex-plored the secretome of HUVECs upon activation by shearstress (10) or with statin treatment (13) thus far. One studyused human microvascular ECs (9), which represent a distinctpopulation of ECs from small vessels. Yet many factors se-creted by ECs were not identified, probably because of theirlow abundance. In this study, we used a secretagogue, phor-bol ester phorbol-12-myristate-13-acetate (PMA) (14, 15), toinduce maximal protein release from serum-starved HUVECsover 45 min. In addition, we applied three different proteomicstrategies for the analysis of glycoproteins/glycopeptides tofurther enrich secreted proteins and characterize their glyco-sylation sites.
EXPERIMENTAL PROCEDURES
EC Culture—HUVECs (Lonza Group Ltd., Basel, Switzerland) werecultured on 0.1% gelatin-coated flasks in M199 medium supple-mented with 1 ng/ml endothelial cell growth factor (Sigma), 3 �g/mlendothelial growth supplement from bovine neural tissue (Sigma), 10U/ml heparin, 1.25 �g/ml thymidine, 10% fetal bovine serum (A15–108, PAA Laboratories, Velizy-Villacoublay, France), and 100 �g/mlpenicillin and streptomycin in a humidified incubator supplementedwith 5% CO2 at 37 °C. The cells were subcultured every 2 to 3 daysat a ratio of 1:4 (16).
Conditioned Medium Collection—HUVECs were cultured in com-plete medium until confluent. Then, they were washed and incubatedin M199 medium for 30 min twice before stimulation with 50 nM PMA(Sigma) in M199 medium for 45 min. The control group was incubatedwith M199 medium in the absence of PMA for 45 min. Conditionedmedia were collected and stored at �80 °C for further analysis.
Immunofluorescence Staining—HUVECs were cultured in Nuncchamber slides (Sigma-Aldrich) for 3 days. HUVECs were stimulated
From ‡The King’s British Heart Foundation Centre, King’s CollegeLondon, London SE5 9NU, UK; §Protein Metrics, San Carlos, CA94070; ¶Thermo Fisher Scientific, Hemel Hempstead, HP2 7GE, UK;�Thermo Fisher Scientific, San Jose, CA 95134
Author’s Choice—Final version full access.Received September 12, 2012, and in revised form, January 18,
2013Published, MCP Papers in Press, January 23, 2013, DOI
10.1074/mcp.M112.024018
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•
Research
Author’s Choice © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.This paper is available on line at http://www.mcponline.org
Molecular & Cellular Proteomics 12.4 1
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with 50 nM PMA in M199 medium for 45 min or incubated with M199medium for 45 min. The cells were fixed with 4% formaldehyde inPBS for 10 min, permeabilized with 0.1% Triton X-100 in PBS for 5min, and blocked in 5% fetal bovine serum in PBS for 30 min at 37 °C.Following 1 h of incubation with the primary antibodies, VE-cadherin(ab33168, Abcam, Cambridge, UK), and von Willebrand factor (vWF)(sc-8068, Santa Cruz Biotechnology, Santa Cruz, CA) at 37 °C, anAlexa Fluor® 594 conjugated donkey anti-rabbit IgG and an AlexaFluor® 488 conjugated donkey anti-goat IgG, respectively, wereadded, and the cells were incubated at 37 °C for 30 min. Nuclei werecounterstained with 4�,6-diamidino-2-phenylindole (D9542, Sigma)for 5 min. The slide was mounted in fluorescence mounting medium(DAKO, Denmark A/S, Glostrup, Denmark) and examined with anAxioPlan 2 fluorescence microscope (Carl Zeiss, Thornwood, NY)(17).
Proteomics Profiling of the Secretome—Conditioned media wereconcentrated with an Amicon spin column (3kD MWCO, EDO Milli-pore Corp., Billerica, MA) and separated via 4%–12% Bis-Tris SDS-PAGE (Invitrogen). Proteins were visualized via silver staining(PlusOne silver staining kit for proteins, GE Healthcare). Gel bandswere digested with modified trypsin (Promega Corp., Madison, WI)overnight on a ProGest digestion robot (Digilab Inc., Marlborough,MA) and analyzed via reverse-phase nano-flow HPLC (PepMap C18,3 �m, 100 Å, 25 cm � 75 �m inner diameter column, ThermoScientific) interfaced to an LTQ Orbitrap XL MS (Thermo Scientific)(18).
Deglycosylation—Concentrated media were mixed with deglyco-sylation buffer (150 mM NaCl, 50 mM sodium acetate, 10 mM EDTA,proteinase inhibitors, pH 6.8) supplemented with 0.05U PNGase F(Sigma), chondroitinase ABC (C3667, Sigma), and keratanase(G6920, Sigma) and incubated at 37 °C overnight (19).
Immunoblotting—Concentrated or deglycosylated media wereseparated via 4%–12% Bis-Tris gel (Invitrogen). Proteins were trans-ferred on a nitrocellulose membrane and blocked with 5% bovineserum albumin in PBS. Membranes were incubated with primaryantibody overnight at 4 °C. Secondary antibodies were incubated for1 h at room temperature. After the addition of ECL (GE Healthcare),the film was developed using a Compact X4 Automatic Processor(Xograph Healthcare Ltd., Stonehouse, UK). The following primaryantibodies were used: agrin (sc-25528, Santa Cruz Biotechnology),biglycan (ab54855, Abcam), connective tissue growth factor (sc-25440, Santa Cruz Biotechnology), fibronectin (sc-56391, Santa CruzBiotechnology), and lymphatic vessel endothelial hyaluronic acid re-ceptor 1 (AF2089, R&D Systems).
Difference Gel Electrophoresis—Conditioned media from HUVECstreated with or without PMA were concentrated using an Amicon spincolumn (3kD MWCO, Millipore) and the ReadyPrep 2D clean-up kit(Bio-Rad). The pellet was resuspended in difference gel electropho-resis lysis buffer (30 mM Tris, 8 M urea, 4% w/v CHAPS, proteaseinhibitors, pH 8.5). For each secretome sample, 15 �g of proteinswere labeled with Cy3 or Cy5. A dye swap was performed to excludepreferential labeling. Cellular extracts of HUVECs were labeled withCy2. Cy2-, Cy3-, and Cy5-labeled samples were separated via iso-electric focusing on immobilized pH gradient dry strips (18 cm, pH3–10 NL, GE Healthcare) with 30 KVH. The strips were equilibratedwith 10 mg/ml DTT in equilibration buffer (6 M urea, 2% w/v SDS, 30%v/v glycerol, 50 mM Tris, pH 8.8) for 15 min followed by 48 mg/mliodoacetamide in equilibration buffer for 15 min before separation viaSDS-PAGE at 100 W for 4 h using an Ettan DALTsix vertical electro-phoresis system (GE Healthcare) (20–22). Gels were scanned on anEttan difference gel electrophoresis imager (GE Healthcare). Imageswere overlaid with ImageQuant TL software (GE Healthcare). Com-mon spots present in both the cellular proteome and the secretomewere excised, digested with trypsin, and identified using nano-flow
HPLC-MS/MS. Detailed protocols are available on our researchgroup’s website.
Glycopeptide Enrichment—Conditioned media were desalted viathe use of Zeba spin columns (Thermo Scientific). Proteins were thenreduced by 5 mM DTT and alkylated with 25 mM iodoacetamide. Afteracetone precipitation overnight, the pellet was resuspended in 100mM triethylammonium bicarbonate (pH 8.5, Sigma) and digested withmodified trypsin (Promega) at 37 °C overnight. Peptides were labeledat a ratio of 100 �g peptides/0.8 mg Tandem Mass Tag Zero (TMT0)(Thermo Scientific) according to the manufacturer’s instruction. La-beled peptides were further enriched for glycopeptides using zwitte-rionic hydrophilic interaction liquid chromatography resin (Merck) (23).
LC/MS of Intact Glycopeptides—The glycopeptide enriched frac-tion was separated using the EASY-nLCTM nano-HPLC system(Thermo Scientific) with a Magic C18 spray tip 15 cm � 75 �m innerdiameter column (Bruker-Michrom, Auburn, CA). Gradient elution wasperformed with 4% to 30% acetonitrile in 0.1% formic acid over 60min at a flow rate of 300 nl/min. The samples were analyzed with anOrbitrap Elite hybrid MS with electron-transfer dissociation (ETD)(Thermo Scientific). The following MS and MS/MS settings were used:Fourier transform: MSn automatic gain control target � 5E4; MS/MS � 1 �scans, max ion time � 200 ms; MS � 300–1800 m/z,resolution � 60,000 at m/z 400, MS target � 1E6; dynamic exclu-sion � repeat count 1, duration 30 s, exclusion duration 90 s; higher-energy C-trap dissociation (HCD): collision energy � 35%, resolu-tion � 15,000; MSn target ion trap � 1E4, 2 �scans, max ion time �150 ms; ETD anion automatic gain control target � 2E5, charge-de-pendent ETD reaction time enabled. For alternating HCD-ETD MS/MS, the top 10 ions were analyzed. For HCD-product-dependentETD, the top 10 ions were analyzed via HCD, and product-dependentETD acquisition was triggered by product (oxonium) ions (m/z163.0812 for Hex; m/z 204.0864 for HexNAc; m/z 138.0554 for Hex-NAc fragment ion) (24).
Deglycosylation with PNGase F and H218O—Zwitterionic hydro-
philic interaction liquid chromatography resin enriched glycopeptideswere resuspended in 50 mM ammonium bicarbonate in H2
18O (97atom % 18O, Sigma) and deglycosylated with PNGase F (Sigma) for4 h at 37 °C. The samples were separated via reverse-phase nano-flow HPLC (PepMap C18, 3 �m, 100 Å, 25 cm � 75 �m innerdiameter column, Thermo Scientific) before analysis on an LTQ Or-bitrap XL MS (Thermo Scientific).
Glycoprotein Enrichment and LC/MS—ConA1 lectin resins (ThermoScientific) were used to enrich glycoproteins from concentrated con-ditioned media according to the manufacturer’s protocol. The input,glycoprotein-enriched fraction, and flow-through samples were sub-jected to trypsin digestion. The in-solution digests were separated ona Thermo Scientific Dionex UltiMate 3000 Rapid Separation LC(RSLC) system using a PepMap C18 column (3 �m, 100 Å, 50 cm �75 �m inner diameter column, Thermo Scientific). The rapid separa-tion LC system was interfaced to a Q Exactive MS (Thermo Scientific),and samples were analyzed using a top-10 HCD method.
Database Search and Data analysis—The following parameterswere used for different experiments.
(i) Gel-LC-MS/MS: Peak lists were generated by Mascot daemon(version 2.3.0, Matrix Science Ltd., London, UK) using extract_msn_com.exe and searched against the UniProt/Swiss-Prot mamma-
1 The abbreviations used are: ConA, concanavalin A; EC, endothe-lial cell; ETD, electron-transfer dissociation; GlcNAc, N-acetylgluco-samine; HCD, higher-energy C-trap dissociation; Hex, hexose;HexNAc, N-acetylhexosamine; HUVEC, human umbilical vein endo-thelial cell; PMA, phorbol-12-myristate-13-acetate; PNGase F, pep-tide: N-glycosidase F; TMT0, Tandem Mass Tag Zero; vWF, vonWillebrand factor.
Glycoproteomics of the Endothelial Secretome
2 Molecular & Cellular Proteomics 12.4
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lian database (version 2012.03, 65,780 entries) using Mascot (version2.3.01, Matrix Science) with peptide tolerance � 10 ppm, MS/MStolerance � 0.8 Da, carbamidomethylation of cysteine as a fixedmodification, oxidation of methionine as a variable modification, anda maximum of two missed cleavage sites. The search results wereloaded into Scaffold software (version 3.6.2, Proteome Software Soft-ware, Inc., Portland, OR). A protein probability greater than 99%, apeptide probability greater than 95%, and a minimum number of twopeptides per protein were applied as filters to generate the protein list.Bovine contaminant proteins are listed separately.
(ii) PNGase F � H218O experiment: Thermo Scientific Proteome
Discoverer software version 1.3 was used to search against theUniProt/Swiss-Prot mammalian database (version 2012.03) usingMascot (version 2.3.01, Matrix Science) with a peptide tolerance of 10ppm; an MS/MS tolerance of 0.8 Da; carbamidomethylation of cys-teine as a fixed modification; oxidation of methionine, TMT0 label onlysine and peptide N-terminus, and deamidation (spontaneousdeamidation in ordinary water) and O18-deamidation (deglycosylationby PNGase F in H2
18O) of asparagine as variable modifications; and amaximum of two missed cleavage sites. Proteome Discoverer pro-duced a custom database containing 136 target proteins based onthis search.
(iii) Orbitrap Elite MS: Raw files were searched against the 136-protein database (along with reversed proteins as decoys) usingByonicTM (25) with a peptide tolerance of 10 ppm; an MS/MS toler-ance of 20 ppm for HCD and 0.6 Da for ETD; the carbamidomethy-lated cysteine, TMT0 label on lysine and peptide N-terminus as fixedmodifications; and oxidation of methionine, deamidation of aspara-gine and glutamine, and phosphorylation of serine and threonine asvariable modifications. ByonicTM allowed one N-glycan modificationon the N-X(not P)-S/T consensus motif per peptide, with mass andcomposition chosen from its “common human” glycan databasecontaining 350 glycan masses up to 6000 Da. Glycan modificationswere verified by the presence of corresponding glycan fragment ions,such as the HexNAc oxonium ion at 204.087 Da in HCD spectra.Peptide sequences were identified by ByonicTM from the ETD spectraand verified manually.
(iv) Q Exactive MS: Raw files were searched against the UniProt/Swiss-Prot human database (version 57.13, 20,266 entries) usingProteome Discoverer (version 1.3, Thermo Scientific) with Mascot(version 2.3.0, Matrix Science) and a peptide tolerance of 10 ppm, anMS/MS tolerance of 10 mmu, carbamidomethylation of cysteine as afixed modification, oxidation of methionine as a variable modification,and a maximum of two missed cleavage sites.
RESULTS
The Secretome of Activated ECs—HUVECs were stimu-lated with PMA, a commonly used secretagogue that inducesexocytosis of endothelial vesicles. As previously reported (26),the morphology of ECs changes from spindle-shaped toround upon PMA activation, and the rod-shaped Weibel-Pal-ade bodies, unique storage vesicles within ECs containingvWF and many other secreted proteins, fuse with the cellmembrane (Fig. 1A). In total, the secretomes of 17 primaryECs were analyzed via gel-LC-MS/MS, with or without degly-cosylation. Apart from 123 secreted proteins, the conditionedmedium of PMA-stimulated ECs was particularly rich in sur-face antigens and receptors, including many established en-dothelial markers (Table I). All identified proteins and peptidesare listed in supplemental Tables S1 and S2, respectively. Thedistribution of the frequencies and the cumulated distribution
of the number of samples in which proteins were identified areshown in supplemental Fig. S1. MS datasets of three biolog-ical replicates have been deposited in PRIDE (accession num-bers 26908–27003).
Immunoblots confirmed that proteins such as fibronectinand biglycan were constitutively secreted (Fig. 1B). Otherssuch as agrin and lymphatic vessel endothelial hyaluronic acidreceptor 1 were released upon PMA stimulation, providing an
FIG. 1. PMA treatment to stimulate EC secretion. Treatment ofHUVECs with PMA, a commonly used secretagogue, resulted in acharacteristic morphological change indicative of activation. A, im-munofluorescence staining of vWF (green) and VE-cadherin (red)shows the exocytotic effect of PMA. B, PMA increased protein se-cretion in the conditioned media as confirmed via immunoblotting. C,relative to previous studies, more than twice as many secreted andplasma membrane proteins were identified. D, overlay of intracellularand secreted proteins by means of difference gel electrophoresis. Inthe left-hand panel, proteins in conditioned media of HUVECs arestained in green (�PMA) and red (�PMA), and cellular proteins arestained in blue. Results were reproduced with different biologicalreplicates using reverse-labeling (right-hand panel: red, �PMA;green, �PMA). The protein corresponding to von Willebrand antigen2 is highlighted with a box. Common proteins in the secretome andthe cellular proteome are numbered in supplemental Fig. S2 andlisted in supplemental Table S3.
Glycoproteomics of the Endothelial Secretome
Molecular & Cellular Proteomics 12.4 3
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LAM
A4
Ext
race
llula
rG
lyco
pro
tein
Lam
inin
sub
unit
bet
a-1
LAM
B1_
HU
MA
NP
0794
2LA
MB
1E
xtra
cellu
lar
Gly
cop
rote
inLa
min
insu
bun
itga
mm
a-1
LAM
C1_
HU
MA
NP
1104
7LA
MC
1E
xtra
cellu
lar
Gly
cop
rote
inLy
sylo
xid
ase
hom
olog
2LO
XL2
_HU
MA
NQ
9Y4K
0LO
XL2
Ext
race
llula
rG
lyco
pro
tein
Mul
timer
in-2
MM
RN
2_H
UM
AN
Q9H
8L6
MM
RN
2E
xtra
cellu
lar
Gly
cop
rote
in
Glycoproteomics of the Endothelial Secretome
4 Molecular & Cellular Proteomics 12.4
tapraid4/zjw-macp/zjw-macp/zjw00413/zjw4413-13a xppws S�4 19/2/13 11:49 4/Color Figure(s) 1–6 ARTNO: M112.024018
TAB
LEI—
cont
inue
d
Pro
tein
nam
eU
niP
rot
IDU
niP
rot
acce
ssio
nnu
mb
erG
ene
nam
eC
ellu
lar
com
pon
ent
Gly
cop
rote
inE
Cm
arke
r
Nid
ogen
-1N
ID1_
HU
MA
NP
1454
3N
ID1
Ext
race
llula
rG
lyco
pro
tein
Nid
ogen
-2N
ID2_
HU
MA
NQ
1411
2N
ID2
Ext
race
llula
rG
lyco
pro
tein
Pro
lyl3
-hyd
roxy
lase
1P
3H1_
HU
MA
NQ
32P
28LE
PR
E1
Ext
race
llula
rG
lyco
pro
tein
Bas
emen
tm
emb
rane
-sp
ecifi
che
par
ansu
lfate
pro
teog
lyca
nco
rep
rote
inP
GB
M_H
UM
AN
P98
160
HS
PG
2E
xtra
cellu
lar
Gly
cop
rote
in
Big
lyca
nP
GS
1_H
UM
AN
P21
810
BG
NE
xtra
cellu
lar
Gly
cop
rote
inP
erox
idas
inho
mol
ogP
XD
N_H
UM
AN
Q92
626
PX
DN
Ext
race
llula
rG
lyco
pro
tein
SP
AR
CS
PR
C_H
UM
AN
P09
486
SP
AR
CE
xtra
cellu
lar
Gly
cop
rote
inTa
rget
ofN
esh-
SH
3TA
RS
H_H
UM
AN
Q7Z
7G0
AB
I3B
PE
xtra
cellu
lar
Gly
cop
rote
inTe
stic
an-1
TIC
N1_
HU
MA
NQ
0862
9S
PO
CK
1E
xtra
cellu
lar
Gly
cop
rote
inTh
rom
bos
pon
din
-1TS
P1_
HU
MA
NP
0799
6TH
BS
1E
xtra
cellu
lar
Pla
sma
mem
bra
neG
lyco
pro
tein
Gro
wth
fact
ors
and
rela
ted
pro
tein
sC
-typ
ele
ctin
dom
ain
fam
ily11
mem
ber
AC
LC11
_HU
MA
NQ
9Y24
0C
LEC
11A
Ext
race
llula
rG
lyco
pro
tein
Cys
tein
e-ric
hm
otor
neur
on1
pro
tein
CR
IM1_
HU
MA
NQ
9NZ
V1
CR
IM1
Ext
race
llula
rP
lasm
am
emb
rane
Gly
cop
rote
inC
onne
ctiv
etis
sue
grow
thfa
ctor
CTG
F_H
UM
AN
P29
279
CTG
FE
xtra
cellu
lar
Gly
cop
rote
inP
rote
inC
YR
61,
insu
lin-l
ike
grow
thfa
ctor
-bin
din
gp
rote
in10
CY
R61
_HU
MA
NO
0062
2C
YR
61E
xtra
cellu
lar
Dic
kkop
f-re
late
dp
rote
in3
DK
K3_
HU
MA
NQ
9UB
P4
DK
K3
Ext
race
llula
rG
lyco
pro
tein
Folli
stat
in-r
elat
edp
rote
in1
FSTL
1_H
UM
AN
Q12
841
FSTL
1E
xtra
cellu
lar
Gly
cop
rote
inH
epat
oma-
der
ived
grow
thfa
ctor
HD
GF_
HU
MA
NP
5185
8H
DG
FE
xtra
cellu
lar
Insu
lin-l
ike
grow
thfa
ctor
-bin
din
gp
rote
in2
IBP
2_H
UM
AN
P18
065
IGFB
P2
Ext
race
llula
rG
lyco
pro
tein
Insu
lin-l
ike
grow
thfa
ctor
-bin
din
gp
rote
in7
IBP
7_H
UM
AN
Q16
270
IGFB
P7
Ext
race
llula
rG
lyco
pro
tein
Late
nt-t
rans
form
ing
grow
thfa
ctor
bet
a-b
ind
ing
pro
tein
1LT
BP
1_H
UM
AN
Q14
766
LTB
P1
Ext
race
llula
rG
lyco
pro
tein
Late
nt-t
rans
form
ing
grow
thfa
ctor
bet
a-b
ind
ing
pro
tein
2LT
BP
2_H
UM
AN
Q14
767
LTB
P2
Ext
race
llula
rG
lyco
pro
tein
Neu
rona
lgro
wth
regu
lato
r1
NE
GR
1_H
UM
AN
Q7Z
3B1
NE
GR
1P
lasm
am
emb
rane
Gly
cop
rote
inIm
mun
ity-
and
infla
mm
atio
n-re
late
dp
rote
ins
Am
yloi
db
eta
A4
pro
tein
A4_
HU
MA
NP
0506
7A
PP
Ext
race
llula
rP
lasm
am
emb
rane
Gly
cop
rote
inB
eta-
2-m
icro
glob
ulin
B2M
G_H
UM
AN
P61
769
B2M
Ext
race
llula
rG
lyco
pro
tein
Com
ple
men
tC
1qtu
mor
necr
osis
fact
or-r
elat
edp
rote
in5
C1Q
T5_H
UM
AN
Q9B
XJ0
C1Q
TNF5
Ext
race
llula
rC
omp
lem
ent
fact
orH
CFA
H_H
UM
AN
P08
603
CFH
Ext
race
llula
rG
lyco
pro
tein
Inte
rleuk
in-2
5,U
PF0
556
pro
tein
C19
orf1
0C
S01
0_H
UM
AN
Q96
9H8
C19
orf1
0E
xtra
cellu
lar
Gra
nulin
sG
RN
_HU
MA
NP
2879
9G
RN
Ext
race
llula
rG
lyco
pro
tein
Inte
rfer
on-i
nduc
edtr
ansm
emb
rane
pro
tein
1IF
M1_
HU
MA
NP
1316
4IF
ITM
1P
lasm
am
emb
rane
Gal
ectin
-1a
LEG
1_H
UM
AN
P09
382
LGA
LS1
Ext
race
llula
rG
alec
tin-3
LEG
3_H
UM
AN
P17
931
LGA
LS3
Ext
race
llula
rM
acro
pha
gem
igra
tion
inhi
bito
ryfa
ctor
aM
IF_H
UM
AN
P14
174
MIF
Ext
race
llula
rN
KG
2Dlig
and
2N
2DL2
_HU
MA
NQ
9BZ
M5
ULB
P2
Ext
race
llula
rP
lasm
am
emb
rane
Gly
cop
rote
inP
entr
axin
-rel
ated
pro
tein
PTX
3P
TX3_
HU
MA
NP
2602
2P
TX3
Ext
race
llula
rG
lyco
pro
tein
Pro
tein
S10
0-A
7S
10A
7_H
UM
AN
P31
151
S10
0A7
Ext
race
llula
rP
rote
inS
100-
A8
S10
A8_
HU
MA
NP
0510
9S
100A
8E
xtra
cellu
lar
Pla
sma
mem
bra
neTu
bul
oint
erst
itial
nep
hriti
san
tigen
-lik
eTI
NA
L_H
UM
AN
Q9G
ZM
7TI
NA
GL1
Ext
race
llula
rG
lyco
pro
tein
Nuc
leas
e-se
nsiti
veel
emen
t-b
ind
ing
pro
tein
1Y
BO
X1_
HU
MA
NP
6780
9Y
BX
1E
xtra
cellu
lar
Zin
c-al
pha
-2-g
lyco
pro
tein
ZA
2G_H
UM
AN
P25
311
AZ
GP
1E
xtra
cellu
lar
Gly
cop
rote
in
Glycoproteomics of the Endothelial Secretome
Molecular & Cellular Proteomics 12.4 5
tapraid4/zjw-macp/zjw-macp/zjw00413/zjw4413-13a xppws S�4 19/2/13 11:49 4/Color Figure(s) 1–6 ARTNO: M112.024018
TAB
LEI—
cont
inue
d
Pro
tein
nam
eU
niP
rot
IDU
niP
rot
acce
ssio
nnu
mb
erG
ene
nam
eC
ellu
lar
com
pon
ent
Gly
cop
rote
inE
Cm
arke
r
Mem
bra
nean
tigen
san
dre
cep
tors
HLA
clas
sI
hist
ocom
pat
ibili
tyan
tigen
,A
-24
alp
hach
ain
1A24
_HU
MA
NP
0553
4H
LA-A
Pla
sma
mem
bra
neG
lyco
pro
tein
HLA
clas
sI
hist
ocom
pat
ibili
tyan
tigen
,A
-30
alp
hach
ain
1A30
_HU
MA
NP
1618
8H
LA-A
Pla
sma
mem
bra
neG
lyco
pro
tein
HLA
clas
sI
hist
ocom
pat
ibili
tyan
tigen
,C
w-1
2al
pha
chai
n1C
12_H
UM
AN
P30
508
HLA
-CP
lasm
am
emb
rane
Gly
cop
rote
in
Alp
ha-2
-mac
rogl
obul
inre
cep
tor-
asso
ciat
edp
rote
inA
MR
P_H
UM
AN
P30
533
LRP
AP
1E
xtra
cellu
lar
Pla
sma
mem
bra
neG
lyco
pro
tein
Bas
alce
llad
hesi
onm
olec
ule
BC
AM
_HU
MA
NP
5089
5B
CA
MP
lasm
am
emb
rane
Gly
cop
rote
inC
omp
lem
ent
com
pon
ent
C1q
rece
pto
rC
1QR
1_H
UM
AN
Q9N
PY
3C
D93
Pla
sma
mem
bra
neG
lyco
pro
tein
EC
mar
ker
Cad
herin
-13
CA
D13
_HU
MA
NP
5529
0C
DH
13P
lasm
am
emb
rane
Gly
cop
rote
inC
adhe
rin-2
CA
DH
2_H
UM
AN
P19
022
CD
H2
Pla
sma
mem
bra
neG
lyco
pro
tein
Cad
herin
-5C
AD
H5_
HU
MA
NP
3315
1C
DH
5P
lasm
am
emb
rane
Gly
cop
rote
inE
Cm
arke
rC
D10
9an
tigen
CD
109_
HU
MA
NQ
6YH
K3
CD
109
Pla
sma
mem
bra
neG
lyco
pro
tein
CD
166
antig
enC
D16
6_H
UM
AN
Q13
740
ALC
AM
Pla
sma
mem
bra
neG
lyco
pro
tein
CD
44an
tigen
CD
44_H
UM
AN
P16
070
CD
44P
lasm
am
emb
rane
Gly
cop
rote
inC
D59
glyc
opro
tein
CD
59_H
UM
AN
P13
987
CD
59E
xtra
cellu
lar
Pla
sma
mem
bra
neG
lyco
pro
tein
CD
9an
tigen
CD
9_H
UM
AN
P21
926
CD
9P
lasm
am
emb
rane
Gly
cop
rote
inC
-typ
ele
ctin
dom
ain
fam
ily14
mem
ber
AC
LC14
_HU
MA
NQ
86T1
3C
LEC
14A
Pla
sma
mem
bra
neG
lyco
pro
tein
Dys
trog
lyca
nD
AG
1_H
UM
AN
Q14
118
DA
G1
Ext
race
llula
rP
lasm
am
emb
rane
Gly
cop
rote
inE
ndog
linE
GLN
_HU
MA
NP
1781
3E
NG
Pla
sma
mem
bra
neG
lyco
pro
tein
EC
mar
ker
End
othe
lialp
rote
inC
rece
pto
rE
PC
R_H
UM
AN
Q9U
NN
8P
RO
CR
Pla
sma
mem
bra
neG
lyco
pro
tein
EC
mar
ker
Ep
hrin
typ
e-B
rece
pto
r4
EP
HB
4_H
UM
AN
P54
760
EP
HB
4P
lasm
am
emb
rane
Gly
cop
rote
inE
ndot
helia
lcel
l-se
lect
ive
adhe
sion
mol
ecul
eE
SA
M_H
UM
AN
Q96
AP
7E
SA
MP
lasm
am
emb
rane
Gly
cop
rote
inE
Cm
arke
rLe
ucin
e-ric
hre
pea
ttr
ansm
emb
rane
pro
tein
FLR
T2FL
RT2
_HU
MA
NO
4315
5FL
RT2
Pla
sma
mem
bra
neG
lyco
pro
tein
Gua
nine
nucl
eotid
e-b
ind
ing
pro
tein
sub
unit
bet
a-2-
like
1aG
BLP
_HU
MA
NP
6324
4G
NB
2L1
Pla
sma
mem
bra
neH
LAcl
ass
Ihi
stoc
omp
atib
ility
antig
en,
alp
hach
ain
EH
LAE
_HU
MA
NP
1374
7H
LA-E
Pla
sma
mem
bra
neG
lyco
pro
tein
Inte
rcel
lula
rad
hesi
onm
olec
ule
1IC
AM
1_H
UM
AN
P05
362
ICA
M1
Ext
race
llula
rP
lasm
am
emb
rane
Gly
cop
rote
inE
Cm
arke
rIn
terc
ellu
lar
adhe
sion
mol
ecul
e2
ICA
M2_
HU
MA
NP
1359
8IC
AM
2P
lasm
am
emb
rane
Gly
cop
rote
inE
Cm
arke
rIn
tegr
inal
pha
-2IT
A2_
HU
MA
NP
1730
1IT
GA
2P
lasm
am
emb
rane
Gly
cop
rote
inIn
tegr
inal
pha
-5IT
A5_
HU
MA
NP
0864
8IT
GA
5P
lasm
am
emb
rane
Gly
cop
rote
inIn
tegr
inal
pha
-6IT
A6_
HU
MA
NP
2322
9IT
GA
6P
lasm
am
emb
rane
Gly
cop
rote
inIn
tegr
inb
eta-
1IT
B1_
HU
MA
NP
0555
6IT
GB
1P
lasm
am
emb
rane
Gly
cop
rote
inE
Cm
arke
rP
rote
inja
gged
-1JA
G1_
HU
MA
NP
7850
4JA
G1
Pla
sma
mem
bra
neG
lyco
pro
tein
Pro
tein
jagg
ed-2
JAG
2_H
UM
AN
Q9Y
219
JAG
2P
lasm
am
emb
rane
Gly
cop
rote
inJu
nctio
nala
dhe
sion
mol
ecul
eA
JAM
1_H
UM
AN
Q9Y
624
F11R
Pla
sma
mem
bra
neG
lyco
pro
tein
BTB
/PO
Zd
omai
n-co
ntai
ning
pro
tein
KC
TD12
KC
D12
_HU
MA
NQ
96C
X2
KC
TD12
Pla
sma
mem
bra
neK
inec
tinK
TN1_
HU
MA
NQ
86U
P2
KTN
1P
lasm
am
emb
rane
Gly
cop
rote
inLy
soso
me-
asso
ciat
edm
emb
rane
glyc
opro
tein
1LA
MP
1_H
UM
AN
P11
279
LAM
P1
Pla
sma
mem
bra
neG
lyco
pro
tein
Low
-den
sity
lipop
rote
inre
cep
tor
LDLR
_HU
MA
NP
0113
0LD
LRP
lasm
am
emb
rane
Gly
cop
rote
inLo
w-d
ensi
tylip
opro
tein
rece
pto
r-re
late
dp
rote
in5
LRP
5_H
UM
AN
O75
197
LRP
5P
lasm
am
emb
rane
Gly
cop
rote
inLy
mp
hatic
vess
elen
dot
helia
lhya
luro
nic
acid
rece
pto
r1
LYV
E1_
HU
MA
NQ
9Y5Y
7LY
VE
1P
lasm
am
emb
rane
Gly
cop
rote
inE
Cm
arke
rH
epat
ocyt
egr
owth
fact
orre
cep
tor
ME
T_H
UM
AN
P08
581
ME
TE
xtra
cellu
lar
Pla
sma
mem
bra
neG
lyco
pro
tein
Cat
ion-
ind
epen
den
tm
anno
se-6
-pho
spha
tere
cep
tor
MP
RI_
HU
MA
NP
1171
7IG
F2R
Pla
sma
mem
bra
neG
lyco
pro
tein
C-t
ype
man
nose
rece
pto
r2
MR
C2_
HU
MA
NQ
9UB
G0
MR
C2
Pla
sma
mem
bra
neG
lyco
pro
tein
Glycoproteomics of the Endothelial Secretome
6 Molecular & Cellular Proteomics 12.4
tapraid4/zjw-macp/zjw-macp/zjw00413/zjw4413-13a xppws S�4 19/2/13 11:49 4/Color Figure(s) 1–6 ARTNO: M112.024018
TAB
LEI—
cont
inue
d
Pro
tein
nam
eU
niP
rot
IDU
niP
rot
acce
ssio
nnu
mb
erG
ene
nam
eC
ellu
lar
com
pon
ent
Gly
cop
rote
inE
Cm
arke
r
Cel
lsur
face
glyc
opro
tein
MU
C18
MU
C18
_HU
MA
NP
4312
1M
CA
MP
lasm
am
emb
rane
Gly
cop
rote
inE
Cm
arke
rN
euro
ligin
-1N
LGN
1_H
UM
AN
Q8N
2Q7
NLG
N1
Pla
sma
mem
bra
neG
lyco
pro
tein
Neu
rona
lcel
lad
hesi
onm
olec
ule
NR
CA
M_H
UM
AN
Q92
823
NR
CA
MP
lasm
am
emb
rane
Gly
cop
rote
inN
euro
pili
n-1
NR
P1_
HU
MA
NO
1478
6N
RP
1E
xtra
cellu
lar
Pla
sma
mem
bra
neG
lyco
pro
tein
Neu
rop
ilin-
2N
RP
2_H
UM
AN
O60
462
NR
P2
Pla
sma
mem
bra
neG
lyco
pro
tein
Neu
rotr
imin
NTR
I_H
UM
AN
Q9P
121
NTM
Pla
sma
mem
bra
neG
lyco
pro
tein
Pro
toca
dhe
rin-1
0P
CD
10_H
UM
AN
Q9P
2E7
PC
DH
10P
lasm
am
emb
rane
Gly
cop
rote
inP
roto
cad
herin
-12
PC
D12
_HU
MA
NQ
9NP
G4
PC
DH
12P
lasm
am
emb
rane
Gly
cop
rote
inP
roto
cad
herin
gam
ma-
A11
PC
DG
B_H
UM
AN
Q9Y
5H2
PC
DH
GA
11P
lasm
am
emb
rane
Gly
cop
rote
inP
roto
cad
herin
gam
ma-
A12
PC
DG
C_H
UM
AN
O60
330
PC
DH
GA
12P
lasm
am
emb
rane
Gly
cop
rote
inP
roto
cad
herin
gam
ma-
B7
PC
DG
J_H
UM
AN
Q9Y
5F8
PC
DH
GB
7P
lasm
am
emb
rane
Gly
cop
rote
inP
roto
cad
herin
-1P
CD
H1_
HU
MA
NQ
0817
4P
CD
H1
Pla
sma
mem
bra
neG
lyco
pro
tein
Pro
toca
dhe
rin-9
PC
DH
9_H
UM
AN
Q9H
C56
PC
DH
9P
lasm
am
emb
rane
Gly
cop
rote
inP
rogr
amm
edce
lld
eath
1lig
and
2P
D1L
2_H
UM
AN
Q9B
Q51
PD
CD
1LG
2E
xtra
cellu
lar
Pla
sma
mem
bra
neG
lyco
pro
tein
Pla
tele
ten
dot
helia
lcel
lad
hesi
onm
olec
ule
PE
CA
1_H
UM
AN
P16
284
PE
CA
M1
Pla
sma
mem
bra
neG
lyco
pro
tein
EC
mar
ker
Ple
xin-
D1
PLX
D1_
HU
MA
NQ
9Y4D
7P
LXN
D1
Pla
sma
mem
bra
neG
lyco
pro
tein
Inac
tive
tyro
sine
-pro
tein
kina
se7
PTK
7_H
UM
AN
Q13
308
PTK
7P
lasm
am
emb
rane
Gly
cop
rote
inR
ecep
tor-
typ
ety
rosi
ne-p
rote
inp
hosp
hata
sed
elta
PTP
RD
_HU
MA
NP
2346
8P
TPR
DP
lasm
am
emb
rane
Gly
cop
rote
inR
ecep
tor-
typ
ety
rosi
ne-p
rote
inp
hosp
hata
seF
PTP
RF_
HU
MA
NP
1058
6P
TPR
FP
lasm
am
emb
rane
Gly
cop
rote
inR
ecep
tor-
typ
ety
rosi
ne-p
rote
inp
hosp
hata
seka
pp
aP
TPR
K_H
UM
AN
Q15
262
PTP
RK
Pla
sma
mem
bra
neG
lyco
pro
tein
Pol
iovi
rus
rece
pto
rP
VR
_HU
MA
NP
1515
1P
VR
Ext
race
llula
rP
lasm
am
emb
rane
Gly
cop
rote
inP
olio
viru
sre
cep
tor-
rela
ted
pro
tein
2P
VR
L2_H
UM
AN
Q92
692
PV
RL2
Pla
sma
mem
bra
neG
lyco
pro
tein
EC
mar
ker
Rou
ndab
out
hom
olog
1R
OB
O1_
HU
MA
NQ
9Y6N
7R
OB
O1
Pla
sma
mem
bra
neG
lyco
pro
tein
Rou
ndab
out
hom
olog
4R
OB
O4_
HU
MA
NQ
8WZ
75R
OB
O4
Pla
sma
mem
bra
neG
lyco
pro
tein
Syn
dec
an-4
SD
C4_
HU
MA
NP
3143
1S
DC
4E
xtra
cellu
lar
Pla
sma
mem
bra
neG
lyco
pro
tein
Sem
apho
rin-4
DS
EM
4D_H
UM
AN
Q92
854
SE
MA
4DP
lasm
am
emb
rane
Gly
cop
rote
inS
emap
horin
-6B
SE
M6B
_HU
MA
NQ
9H3T
3S
EM
A6B
Pla
sma
mem
bra
neG
lyco
pro
tein
Tyro
sine
-pro
tein
pho
spha
tase
non-
rece
pto
rty
pe
sub
stra
te1
SH
PS
1_H
UM
AN
P78
324
SIR
PA
Pla
sma
mem
bra
neG
lyco
pro
tein
Sta
bili
n-1
STA
B1_
HU
MA
NQ
9NY
15S
TAB
1P
lasm
am
emb
rane
Gly
cop
rote
inE
Cm
arke
rTr
ansf
errin
rece
pto
rp
rote
in1
TFR
1_H
UM
AN
P02
786
TFR
CE
xtra
cellu
lar
Pla
sma
mem
bra
neG
lyco
pro
tein
Tyro
sine
-pro
tein
kina
sere
cep
tor
Tie-
1TI
E1_
HU
MA
NP
3559
0TI
E1
Pla
sma
mem
bra
neG
lyco
pro
tein
Tyro
sine
-pro
tein
kina
sere
cep
tor
UFO
UFO
_HU
MA
NP
3053
0A
XL
Ext
race
llula
rP
lasm
am
emb
rane
Gly
cop
rote
inV
ascu
lar
end
othe
lialg
row
thfa
ctor
rece
pto
r2
VG
FR2_
HU
MA
NP
3596
8K
DR
Ext
race
llula
rP
lasm
am
emb
rane
Gly
cop
rote
inE
Cm
arke
rV
ascu
lar
end
othe
lialg
row
thfa
ctor
rece
pto
r3
VG
FR3_
HU
MA
NP
3591
6FL
T4E
xtra
cellu
lar
Pla
sma
mem
bra
neG
lyco
pro
tein
EC
mar
ker
Ver
ylo
w-d
ensi
tylip
opro
tein
rece
pto
rV
LDLR
_HU
MA
NP
9815
5V
LDLR
Pla
sma
mem
bra
neG
lyco
pro
tein
Mis
cella
neou
sm
emb
rane
pro
tein
sB
rain
acid
solu
ble
pro
tein
1B
AS
P1_
HU
MA
NP
8072
3B
AS
P1
Pla
sma
mem
bra
neD
naJ
hom
olog
sub
fam
ilyB
mem
ber
4D
NJB
4_H
UM
AN
Q9U
DY
4D
NA
JB4
Pla
sma
mem
bra
neR
NA
-bin
din
gp
rote
inE
WS
EW
S_H
UM
AN
Q01
844
EW
SR
1P
lasm
am
emb
rane
Nck
-ass
ocia
ted
pro
tein
1N
CK
P1_
HU
MA
NQ
9Y2A
7N
CK
AP
1P
lasm
am
emb
rane
Na(
�)/
H(�
)ex
chan
gere
gula
tory
cofa
ctor
NH
E-R
F2N
HR
F2_H
UM
AN
Q15
599
SLC
9A3R
2P
lasm
am
emb
rane
Pol
ymer
ase
Ian
dtr
ansc
ript
rele
ase
fact
orP
TRF_
HU
MA
NQ
6NZ
I2P
TRF
Pla
sma
mem
bra
neS
erum
dep
rivat
ion-
resp
onse
pro
tein
SD
PR
_HU
MA
NO
9581
0S
DP
RP
lasm
am
emb
rane
Sus
hire
pea
t-co
ntai
ning
pro
tein
SR
PX
2S
RP
X2_
HU
MA
NO
6068
7S
RP
X2
Ext
race
llula
rE
ryth
rocy
teb
and
7in
tegr
alm
emb
rane
pro
tein
STO
M_H
UM
AN
P27
105
STO
MP
lasm
am
emb
rane
Glycoproteomics of the Endothelial Secretome
Molecular & Cellular Proteomics 12.4 7
tapraid4/zjw-macp/zjw-macp/zjw00413/zjw4413-13a xppws S�4 19/2/13 11:49 4/Color Figure(s) 1–6 ARTNO: M112.024018
TAB
LEI—
cont
inue
d
Pro
tein
nam
eU
niP
rot
IDU
niP
rot
acce
ssio
nnu
mb
erG
ene
nam
eC
ellu
lar
com
pon
ent
Gly
cop
rote
inE
Cm
arke
r
Mis
cella
neou
sse
cret
edp
rote
ins
Pep
tidyl
-gly
cine
alp
ha-a
mid
atin
gm
onoo
xyge
nase
AM
D_H
UM
AN
P19
021
PA
ME
xtra
cellu
lar
Gly
cop
rote
inA
ngio
poi
etin
-2A
NG
P2_
HU
MA
NO
1512
3A
NG
PT2
Ext
race
llula
rG
lyco
pro
tein
End
othe
lin-1
ED
N1_
HU
MA
NP
0530
5E
DN
1E
xtra
cellu
lar
End
othe
lialc
ell-
spec
ific
mol
ecul
e1
ES
M1_
HU
MA
NQ
9NQ
30E
SM
1E
xtra
cellu
lar
Gly
cop
rote
inP
rote
inFA
M3C
FAM
3C_H
UM
AN
Q92
520
WN
T16
Ext
race
llula
rE
pid
idym
alse
cret
ory
pro
tein
E1
NP
C2_
HU
MA
NP
6191
6N
PC
2E
xtra
cellu
lar
Gly
cop
rote
inP
rogr
amm
edce
lld
eath
pro
tein
10P
DC
10_H
UM
AN
Q9B
UL8
PD
CD
10P
lasm
am
emb
rane
Pro
lact
in-i
nduc
ible
pro
tein
PIP
_HU
MA
NP
1227
3P
IPE
xtra
cellu
lar
Gly
cop
rote
inS
ulfh
ydry
loxi
das
e1
QS
OX
1_H
UM
AN
O00
391
QS
OX
1E
xtra
cellu
lar
Gly
cop
rote
inS
ecre
togl
obin
fam
ily1D
mem
ber
2S
G1D
2_H
UM
AN
O95
969
SC
GB
1D2
Ext
race
llula
rTh
iore
dox
ina
THIO
_HU
MA
NP
1059
9TX
NE
xtra
cellu
lar
Thym
osin
bet
a-4
TYB
4_H
UM
AN
P62
328
TMS
B4X
Ext
race
llula
rP
rote
ase
inhi
bito
rsC
ysta
tin-C
CY
TC_H
UM
AN
P01
034
CS
T3E
xtra
cellu
lar
Gly
cop
rote
inLe
ukoc
yte
elas
tase
inhi
bito
rIL
EU
_HU
MA
NP
3074
0S
ER
PIN
B1
Ext
race
llula
rIn
ter-
alp
ha-t
ryp
sin
inhi
bito
rhe
avy
chai
nH
2IT
IH2_
HU
MA
NP
1982
3IT
IH2
Ext
race
llula
rG
lyco
pro
tein
Ser
pin
B9
SP
B9_
HU
MA
NP
5045
3S
ER
PIN
B9
Ext
race
llula
rM
etal
lop
rote
inas
ein
hib
itor
1TI
MP
1_H
UM
AN
P01
033
TIM
P1
Ext
race
llula
rG
lyco
pro
tein
Met
allo
pro
tein
ase
inhi
bito
r2
TIM
P2_
HU
MA
NP
1603
5TI
MP
2E
xtra
cellu
lar
Pro
teas
esA
ngio
tens
in-c
onve
rtin
gen
zym
eA
CE
_HU
MA
NP
1282
1A
CE
Ext
race
llula
rP
lasm
am
emb
rane
Gly
cop
rote
inE
Cm
arke
rD
isin
tegr
inan
dm
etal
lop
rote
inas
ed
omai
n-co
ntai
ning
pro
tein
10A
DA
10_H
UM
AN
O14
672
AD
AM
10P
lasm
am
emb
rane
Gly
cop
rote
in
Am
inop
eptid
ase
BA
MP
B_H
UM
AN
Q9H
4A4
RN
PE
PE
xtra
cellu
lar
Am
inop
eptid
ase
NA
MP
N_H
UM
AN
P15
144
AN
PE
PP
lasm
am
emb
rane
Gly
cop
rote
inB
one
mor
pho
gene
ticp
rote
in1
BM
P1_
HU
MA
NP
1349
7B
MP
1E
xtra
cellu
lar
Gly
cop
rote
inC
athe
psi
nB
CA
TB_H
UM
AN
P07
858
CTS
BE
xtra
cellu
lar
Gly
cop
rote
inC
athe
psi
nD
CA
TD_H
UM
AN
P07
339
CTS
DE
xtra
cellu
lar
Gly
cop
rote
inC
athe
psi
nZ
CA
TZ_H
UM
AN
Q9U
BR
2C
TSZ
Ext
race
llula
rG
lyco
pro
tein
Car
box
ypep
tidas
eQ
CB
PQ
_HU
MA
NQ
9Y64
6C
PQ
Ext
race
llula
rG
lyco
pro
tein
Dip
eptid
ylp
eptid
ase
2D
PP
2_H
UM
AN
Q9U
HL4
DP
P7
Ext
race
llula
rG
lyco
pro
tein
Dip
eptid
ylp
eptid
ase
3D
PP
3_H
UM
AN
Q9N
Y33
DP
P3
Pla
sma
mem
bra
neE
ndop
lasm
icre
ticul
umam
inop
eptid
ase
1E
RA
P1_
HU
MA
NQ
9NZ
08E
RA
P1
Ext
race
llula
rG
lyco
pro
tein
Furin
FUR
IN_H
UM
AN
P09
958
FUR
INP
lasm
am
emb
rane
Gly
cop
rote
inG
amm
a-gl
utam
ylhy
dro
lase
GG
H_H
UM
AN
Q92
820
GG
HE
xtra
cellu
lar
Gly
cop
rote
inS
erin
ep
rote
ase
HTR
A1
HTR
A1_
HU
MA
NQ
9274
3H
TRA
1E
xtra
cellu
lar
Insu
lin-d
egra
din
gen
zym
eID
E_H
UM
AN
P14
735
IDE
Ext
race
llula
rP
lasm
am
emb
rane
Inte
rstit
ialc
olla
gena
seM
MP
1_H
UM
AN
P03
956
MM
P1
Ext
race
llula
rG
lyco
pro
tein
Str
omel
ysin
-2M
MP
10_H
UM
AN
P09
238
MM
P10
Ext
race
llula
rM
atrix
met
allo
pro
tein
ase-
14M
MP
14_H
UM
AN
P50
281
MM
P14
Pla
sma
mem
bra
ne72
kDa
typ
eIV
colla
gena
seM
MP
2_H
UM
AN
P08
253
MM
P2
Ext
race
llula
rG
lyco
pro
tein
Lyso
som
alP
ro-X
carb
oxyp
eptid
ase
PC
P_H
UM
AN
P42
785
PR
CP
Pla
sma
mem
bra
neG
lyco
pro
tein
Ser
ine
pro
teas
e23
PR
S23
_HU
MA
NO
9508
4P
RS
S23
Ext
race
llula
rG
lyco
pro
tein
Ub
iqui
tinca
rbox
yl-t
erm
inal
hyd
rola
se14
UB
P14
_HU
MA
NP
5457
8U
SP
14P
lasm
am
emb
rane
Glycoproteomics of the Endothelial Secretome
8 Molecular & Cellular Proteomics 12.4
tapraid4/zjw-macp/zjw-macp/zjw00413/zjw4413-13a xppws S�4 19/2/13 11:49 4/Color Figure(s) 1–6 ARTNO: M112.024018
TAB
LEI—
cont
inue
d
Pro
tein
nam
eU
niP
rot
IDU
niP
rot
acce
ssio
nnu
mb
erG
ene
nam
eC
ellu
lar
com
pon
ent
Gly
cop
rote
inE
Cm
arke
r
Sig
nalt
rans
duc
tion
pro
tein
sA
den
ylyl
cycl
ase-
asso
ciat
edp
rote
in1
CA
P1_
HU
MA
NQ
0151
8C
AP
1P
lasm
am
emb
rane
Cel
ldiv
isio
nco
ntro
lpro
tein
42ho
mol
ogC
DC
42_H
UM
AN
P60
953
CD
C42
Pla
sma
mem
bra
neC
onta
ctin
-ass
ocia
ted
pro
tein
-lik
e3
CN
TP3_
HU
MA
NQ
9BZ
76C
NTN
AP
3E
xtra
cellu
lar
Pla
sma
mem
bra
neG
lyco
pro
tein
Ad
apte
rm
olec
ule
crk
CR
K_H
UM
AN
P46
108
CR
KP
lasm
am
emb
rane
Ras
GTP
ase-
activ
atin
gp
rote
in-b
ind
ing
pro
tein
1G
3BP
1_H
UM
AN
Q13
283
G3B
P1
Pla
sma
mem
bra
neG
row
thar
rest
-sp
ecifi
cp
rote
in6
GA
S6_
HU
MA
NQ
1439
3G
AS
6E
xtra
cellu
lar
Gly
cop
rote
inIn
terf
eron
-ind
uced
guan
ylat
e-b
ind
ing
pro
tein
1G
BP
1_H
UM
AN
P32
455
GB
P1
Ext
race
llula
rG
uani
nenu
cleo
tide-
bin
din
gp
rote
inG
(i)su
bun
ital
pha
-2G
NA
I2_H
UM
AN
P04
899
GN
AI2
Pla
sma
mem
bra
neG
lyp
ican
-1G
PC
1_H
UM
AN
P35
052
GP
C1
Ext
race
llula
rP
lasm
am
emb
rane
Gly
cop
rote
inH
edge
hog-
inte
ract
ing
pro
tein
HH
IP_H
UM
AN
Q96
QV
1H
HIP
Ext
race
llula
rP
lasm
am
emb
rane
Gly
cop
rote
inH
istid
ine
tria
dnu
cleo
tide-
bin
din
gp
rote
in1a
HIN
T1_H
UM
AN
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Glycoproteomics of the Endothelial Secretome
Molecular & Cellular Proteomics 12.4 9
tapraid4/zjw-macp/zjw-macp/zjw00413/zjw4413-13a xppws S�4 19/2/13 11:49 4/Color Figure(s) 1–6 ARTNO: M112.024018
explanation for why previously unidentified proteins (8, 10)were found in the present analysis (Fig. 1C). An overlay be-tween secreted (Cy3 and Cy 5; green and red color) andcellular (Cy 2; blue color) proteins is shown in Fig. 1D. Com-mon spots were numbered (supplemental Fig. S2) and iden-tified via LC-MS/MS (supplemental Table S3). Certain pro-teins, such as von Willebrand antigen 2 (a propeptide of vWF,AA 23–763), were clearly more abundant in the secretome ofPMA-treated HUVECs.
The Endothelial Glycoproteome—Among the 1252 identi-fied proteins were 253 extracellular or plasma membraneproteins (approximately 20%) related to cell adhesion, bloodcoagulation, hemostasis, signaling transduction, and proteintransportation, of which 166 were known glycoproteins (TableI). To further characterize this subproteome, we employed aglycoproteomics approach. Secreted proteins were precipi-tated and digested with trypsin, and tryptic peptides werelabeled with TMT0 to increase their charge state prior toenrichment by means of zwitterionic hydrophilic interactionliquid chromatography purification (24). For glycosite identifi-cation, an indirect and a direct strategy were pursued (Fig.2A): (i) digestion with PNGase F in the presence of 18O waterto label the conversion of asparagine to aspartic acid upon theremoval of N-glycans, and (ii) alternating HCD and ETD (HCD-
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18O treatment, re-spectively: identified unique glycopeptides (B), unique glycosylationsites (C), and unique glycoproteins (D).
Glycoproteomics of the Endothelial Secretome
10 Molecular & Cellular Proteomics 12.4
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tapraid4/zjw-macp/zjw-macp/zjw00413/zjw4413-13a xppws S�4 19/2/13 11:49 4/Color Figure(s) 1–6 ARTNO: M112.024018
alt-ETD) or HCD-product-dependent ETD (HCD-pd-ETD)fragmentation on an Orbitrap Elite MS (24).
There was little overlap in the numbers of glycopeptides(Fig. 2B) and glycosylation sites (Fig. 2C) identified via thedirect (HCD-ETD) and the indirect (PNGase F � H2
18O) meth-ods. Better agreement was observed at the protein level (Fig.2D). With the indirect (PNGase F � H2
18O) method, 27 pep-tides were identified with N[�2.99] modification at non-con-sensus sequence, out of 1139 total identified peptides withN[�2.99]. This anomaly rate of 2.4% (27/1139) combines therate of false identifications and the rate of chance deamida-tions in 18O water that were not in the consensus sequence ofglycosylation (i.e. N-X(not P)-S/T). All glycopeptides identifiedare listed in Table II and supplemental Table S4. Three spectra(full MS, HCD, and ETD) from a neuronal cell adhesion mole-cule (UniProt accession number Q92823) (AA - 222FNHTQ-TIQQK231) are presented in Fig. 3.
For the same samples, HCD-pd-ETD revealed 28 known,25 potential, and 16 novel glycosylation sites based on 209identified spectra; HCD-alt-ETD revealed 20 known, 32 po-tential, and 14 novel glycosylation sites from 110 identifiedspectra. The HCD-alt-ETD method selected mostly precur-sors with higher intensities, higher charge, and smaller m/z(Fig. 4A). Several large glycopeptides were detected via onlyHCD-alt-ETD, and more low-abundant glycopeptides weredetected via HCD-pd-ETD. There was limited overlap in theidentified glycopeptides but better agreement in the proteinlevel (Fig. 4B). Among the 319 total glycopeptides identified inthe conditioned media, 31 were attached with a trimannosylcore (-HexNAc2Hex3) or truncated core (-HexNAc2Hex), 50with high mannose (-HexNAc2Hex4–9), and 238 with complex/hybrid glycans. Notably, HCD-pd-ETD detected almost twiceas many complex/hybrid glycoforms as HCD-alt-ETD(Fig.4C).
Validation of Glycoproteins—To validate the glycosylationstatus, we performed additional analysis before and afterglycoprotein enrichment with affinity resins of ConA lectin(n � 4) using a Q Exactive MS (Thermo Scientific). We thencompared the number of identified spectra in the glycopro-tein-enriched fraction, the flow-through, and the input (sup-plemental Table S5). For most glycoproteins, a higher spectralcount was observed in the glycoprotein-enriched fractionthan in the original input and/or the flow-through. Represent-ative examples (fibronectin, neuronal cell adhesion molecule,tyrosine-protein-kinase-like 7, and vWF) are shown in Fig. 5A.Non-glycosylated proteins, such as annexin A2 and alpha-enolase, were more abundant in the flow-through. Glycopro-teins identified in all three methods are highlighted in Fig. 5B.
Confirmation of Predicted Glycosylation Sites—The hemo-static protein vWF is the main protein stored within Weibel-Palade bodies (27). After secretagogue stimulation, Weibel-Palade bodies undergo exocytosis, releasing vWF filaments.vWF is one of the few known proteins containing the ABOblood group signature, which is formed by different glycans.
Although the released glycan composition of this protein hasbeen investigated extensively (28, 29), experimental evidencefor many putative glycosylation sites is still missing. The cov-erage obtained for vWF in our proteomics analysis is shown inFig. 6A. The precursor protein consists of homologous unitssuch as the VWF type A, C, and D domains and a C-terminalcystine know (CTCK). The vWF propeptide (D1-D2, AA 23–763) is separated from the remaining domains of mature vWF(AA 764–2813) via furin-mediated proteolytic cleavage. Weconfirmed 6 N-glycosylation sites. Notably, three N-glycosy-lation sites were located within the propeptide (AA 23–763).Examples of ETD spectra are shown in Fig. 6B.
DISCUSSION
This study represents a significant advance over the exist-ing proteomics literature on ECs. Unlike other cell types, ECsdo not tolerate prolonged serum starvation, and their suscep-tibility to cell death upon serum withdrawal poses a majorchallenge for proteomic workflows targeting their secretome.We performed secretome analysis after 45 min of PMA stim-ulation combined with enrichment strategies for glycopro-teins and glycopeptides. Glycopeptides were analyzed viathree complementary MS techniques: the detection of 18Oasparagine deamidation after digestion with PNGase F inH2
18O, HCD-alt-ETD, and HCD-pd-ETD using an OrbitrapElite MS.
The Endothelial Secretome—The secretagogue PMA mini-mized EC death by allowing a shorter incubation period underserum-free conditions while increasing coverage in the pro-teomic analysis by inducing the exocytosis of intracellularstorage vesicles (14) such as Weibel-Palade bodies. Theseunique storage vesicles in ECs play a major role in hemostasisand cell-to-cell communication. Using this approach, manymore proteins were identified than in any previous proteomicsstudy on ECs, including known endothelial surface markerssuch as endoglin (CD105), integrin beta-1 (CD29), tyrosine-protein kinase receptor Tie-1, and junctional adhesion mole-cule A; secreted growth factors (i.e. C-type lectin domainfamily 11 member A); co-receptors (i.e. neuropilin-1 (co-re-ceptor for VEGF-A)); proteases(i.e. furin); and inflammatorymediators (i.e. macrophage migration inhibitory factor), toname just a few. Short-term PMA treatment does not releasemicroparticles (30), as shedding events make it difficult todiscern intracellular from secreted/membrane proteins. In adirect comparison of the cellular proteome and the secretomeutilizing difference gel electrophoresis, 70 out of 96 proteinsanalyzed were present in both samples, representing �10%of the visible protein spots in the secretome.
Biological Importance of Glycosylation—Glycosylation iskey for the stability and solubility of secreted and membraneproteins. It is the most complex post-translational modifica-tion (31) and mediates extracellular matrix network assembly,cell–cell interactions, and cell–matrix interactions. Unlikepolynucleotides and polypeptides, which have a linear struc-
Glycoproteomics of the Endothelial Secretome
Molecular & Cellular Proteomics 12.4 11
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Glycoproteomics of the Endothelial Secretome
12 Molecular & Cellular Proteomics 12.4
tapraid4/zjw-macp/zjw-macp/zjw00413/zjw4413-13a xppws S�4 19/2/13 11:49 4/Color Figure(s) 1–6 ARTNO: M112.024018
TAB
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9
Glycoproteomics of the Endothelial Secretome
Molecular & Cellular Proteomics 12.4 13
tapraid4/zjw-macp/zjw-macp/zjw00413/zjw4413-13a xppws S�4 19/2/13 11:49 4/Color Figure(s) 1–6 ARTNO: M112.024018
TAB
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14 Molecular & Cellular Proteomics 12.4
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Glycoproteomics of the Endothelial Secretome
Molecular & Cellular Proteomics 12.4 15
tapraid4/zjw-macp/zjw-macp/zjw00413/zjw4413-13a xppws S�4 19/2/13 11:49 4/Color Figure(s) 1–6 ARTNO: M112.024018
TAB
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Glycoproteomics of the Endothelial Secretome
16 Molecular & Cellular Proteomics 12.4
tapraid4/zjw-macp/zjw-macp/zjw00413/zjw4413-13a xppws S�4 19/2/13 11:49 4/Color Figure(s) 1–6 ARTNO: M112.024018
ture, sugars tend to be arranged in branched polymers, re-sulting in an exponential increase of possible polysaccharidecombinations. Theoretically, just six monosaccharides can
give rise to 1012 different glycan structures. This high diversityof protein-bound glycans requires a combination of differenttechniques. For example, new MS-based methods were de-
FIG. 3. HCD-pd-ETD fragmentation. Full MS showing the different glycoforms of the same peptide sequence (A). Characteristic oxoniumion detected by HCD at m/z � 204.09 (B). This HexNAc signature triggered an ETD scan to identify the peptide sequence and confirm theglycosylation site (C).
Glycoproteomics of the Endothelial Secretome
Molecular & Cellular Proteomics 12.4 17
COLOR
tapraid4/zjw-macp/zjw-macp/zjw00413/zjw4413-13a xppws S�4 19/2/13 11:49 4/Color Figure(s) 1–6 ARTNO: M112.024018
veloped to profile the cell surface N-glycoproteome as adifferentiation marker for stem cells (32). We applied a com-bination of different glycoproteomics techniques to furtherenrich for secreted and shed membrane proteins and revealpotential glycosylation sites within the endothelial secretome.Glycoproteins play important roles in many biological pro-cesses related to ECs, such as angiogenesis, in which thestructural change of the glycans will determine the attachment
property of cells and influence cell-to-cell interactions (33).Interestingly, vWF is a glycoprotein produced uniquely by ECsand megakaryocytes. Previous publications investigatingvWF isolated from plasma failed to identify glycosylation siteswithin the propeptide (29). In plasma, the concentration of thepropeptide is about one-tenth of the concentration of maturevWF (34, 35). In the conditioned medium of ECs, however, weobserved several glycopeptides of the propeptide. Thus, the
FIG. 4. Comparison of HCD-pd-ETD and HCD-alt-ETD. The two methods, HCD-pd-ETD (blue) and HCD-alt-ETD (red), displayed distinctdistributions of the observed m/z, charge state, mass of identified peptides (M�H), and glycan mass, as well as the intensity of the precursorions and the ByonicsTM score (all y-axes). The x-axes represent index numbers after proteins were sorted by their corresponding y-axis valuefrom lower to higher (A). There was limited overlap in the identified glycopeptides (B). C, the HCD-pd-ETD method preferentially identifiedcomplex/hybrid glycans.
Glycoproteomics of the Endothelial Secretome
18 Molecular & Cellular Proteomics 12.4
COLOR
tapraid4/zjw-macp/zjw-macp/zjw00413/zjw4413-13a xppws S�4 19/2/13 11:49 4/Color Figure(s) 1–6 ARTNO: M112.024018
endothelial secretome allowed us to interrogate the glycosy-lation sites of von Willebrand antigen 2, the N-terminal cleav-age product of vWF that aids N-terminal multimerization andprotein compartmentalization of mature vWF in storagegranules.
Conventional Methods for Glycoproteomics—As reviewedelsewhere (36), conventional glycoproteomic methods involvethe enrichment of glycoproteins (typically with lectins likeConA and wheat germ agglutinin), cleavage of the glycans,and identification of the remaining peptide sequence. Themost widely used method for detecting N-glycopeptides isdigestion by PNGase F. PNGase F cleaves the GlcNAc mol-
ecule closest to the peptide (37). After PNGase F treatment,formerly N-linked glycosylated peptides are identified basedon the conversion of Asn to Asp (deamidation) in the consen-sus motif for N-linked glycosylation (sequence N-X(not P)-S/T). This method has two major caveats. The first of these is ahigh false positive rate due to spontaneous deamidation. Asn-Gly sites, in particular, are prone to spontaneous deamidation(38–40). To reduce false positives, PNGase F treatment isperformed in 18O water, adding a larger tag of 2.99 Da.Importantly, all known glycosyltransferases that mediate N-linked glycosylation are supposed to recognize a consensusmotif, and this consensus sequence for N-linked glycosylation
FIG. 5. Glycoprotein enrichment for validation. A, spectral count of input, glycoprotein-enriched fraction (GP), and flow-through fraction(FT) from representative glycoproteins and non-glycoproteins. B, complementarity of the different methods (HCD-ETD, PNGase F � H2
18Otreatment, and glycoprotein enrichment). Only 18 glycoproteins were consistently identified.
Glycoproteomics of the Endothelial Secretome
Molecular & Cellular Proteomics 12.4 19
COLOR
tapraid4/zjw-macp/zjw-macp/zjw00413/zjw4413-13a xppws S�4 19/2/13 11:49 4/Color Figure(s) 1–6 ARTNO: M112.024018
FIG. 6. Sequence coverage for vWF. A, schematic illustration of vWF sequence. Coverage is highlighted in green, and potentialglycosylation sites are shown in red. A large hexagon indicates a glycosylation site with a reference in the Uniprot database. By using theHCD-ETD (H) or PNGase F (P) method, we confirmed six N-glycosylation sites on vWF. B, ETD spectra of glycopeptides identified via HCD-ETD(N156, N211, N666, N1574). The following abbreviations are used: a, y, g, k � TMT modified Ala, Tyr, Gly, and Lys, respectively; c �carboxyamidomethylation of Cys; m � oxidation of Met.
Glycoproteomics of the Endothelial Secretome
20 Molecular & Cellular Proteomics 12.4
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must be taken into consideration (41). 2) The second caveat isthat after PNGase F cleavage, the released sugars can beanalyzed separately, but the link to the identified peptideswith deamidated amino acids is lost (42, 43). Ideally, intactglycopeptides are analyzed directly via MS/MS even in com-plex biological samples.
Novel HCD-ETD Method—HCD fragmentation mostlybreaks glycosidic bonds, whereas ETD preserves the glycanattachment and fragments the peptide backbone, providingmore complete peptide sequence information. CurrentMS/MS acquisition strategies for glycopeptide analysis relyon the acquisition of MS/MS spectra for all precursor ions. Inthis study, HCD was employed to generate glycan oxoniumions and trigger an ETD spectrum in a data-dependent man-ner. HCD presents the sugar signatures within the low m/zrange, which are otherwise lost as a result of the one-third ruleof ion trap fragmentation (44). Glycopeptides with terminalHexNAc generate typically an m/z 204.0864 oxonium ion andits fragments at m/z 168.0653 and 138.0550. The oxonium ionand its fragments are measured with the high mass accuracyof the Orbitrap analyzer, and the unambiguous identificationof the glycan oxonium ion generated by the HCD scan servesas a diagnostic marker for glycopeptides. This approach wascompared against conventional HCD-alt-ETD scans using acomplex biological sample. The HCD-alt-ETD preferentiallydetects higher charged and higher intensity precursor ionsthan HCD-pd-ETD. This might be because (i) a higher chargeincreases ETD fragmentation efficiency, resulting in moreidentified glycopeptides; (ii) high-charged precursors did notproduce HCD spectra of sufficient quality to trigger ETDbased on the diagnostic oxonium ions; or (iii) more abundantpeptides were selected in HCD-alt-ETD because the instru-ment duty cycle is less efficient than in HCD-pd-ETD. Overall,the combination of multiple MS methods used in our studyprovides greater confidence in the identification of glycopep-tides than studies relying on a single approach and offerscomplementary advantages in the assessment of the glyco-proteome, notably, the simultaneous identification of the pep-tide sequence, the glycosylation site, and the glycancomposition.
Study Limitations—N-linked and O-linked glycosylation arethe two most common forms of glycosylation in mammals(45). Only N-linked glycosylation was analyzed in the presentstudy. Unlike N-linked glycosylation, O-linked glycosylationhas no consensus site (46). This makes the analysis of O-linked glycopeptides a more daunting task (47). Lectins arewidely used for glycoprotein enrichment. There are manytypes of lectins binding to different sugars, such as ConA(binds to �-D-mannosyl and �-D-glucosyl residues) and wheatgerm agglutinin (binds to GlcNAc�1–4GlcNAc�1–4GlcNAc-and N-acetylneuraminic acid). Here we used only ConA as aproof of principle to demonstrate the complementary resultsof multiple glycoprotein identification methods. ConA isknown to display nonspecific avidity for hydrophobic ligands
such as certain domains of tropomyosin (48). Furthermore,the standard protocol for the ConA glycoprotein enrichmentkit is not optimized for cleanliness, and several known non-glycoproteins were also detected in the eluate samples. Se-quential washes with low- and high-ionic-strength buffersbefore elution might have reduced this contamination (49).Also, mixing different lectins would increase the coverage ofthe glycoproteome in biological samples (39). Additional ef-forts are needed for a complete structural characterization ofprotein glycosylation; in particular, the quantitation of theoccupancy rates and the identification of the glycan structureas complex/hybrid glycans cannot be discerned via our cur-rent MS approach.
CONCLUSIONS
Cardiovascular diseases arise from exposure to risk factorsthat induce complex pathophysiological perturbations of en-dothelial protein secretion. The recent advent of new pro-teomic technologies has enabled us to obtain information onthe dynamic regulation of endothelial protein secretion. Wepresent results from an extensive glycoproteomic analysiswith information on glycan composition obtained via a directMS method. Future proteomics studies linking endothelialsecretory processes to cardiovascular risk factors and endo-thelial dysfunction will provide valuable insights about themechanisms contributing to cardiovascular disease.
Acknowledgment—We thank Dr. Sarah Langley for assistance withthe gene ontology annotation.
* This work was funded by the Department of Health via a NationalInstitute for Health Research (NIHR) Biomedical Research Centreaward to Guy’s and St. Thomas’ NHS Foundation Trust in partnershipwith King’s College London and King’s College Hospital NHS Foun-dation Trust. Dr. M. Mayr is supported by a Senior Research Fellow-ship of the British Heart Foundation.
□S This article contains supplemental material.** To whom correspondence should be addressed: Prof. Manuel
Mayr, Cardiovascular Division, King’s British Heart Foundation Cen-tre, King’s College London, 125 Coldharbour Lane, London SE5 9NU,UK, Tel.: �44 (0) 20 7848 5132, E-mail: [email protected].
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