The proteomic profiling of calenduloside e targets in HUVEC: Design, synthesis and application of biotinylated probe BCEA

Article abstract of DOI:10.1039/c6ra25572h

We previously found the total saponins from the medicinal herb Aralia elata, which exhibited strong anti-oxidative and anti-apoptotic effects. calenduloside E (CE) is one of the major triterpenoid saponin compounds isolated from these total saponins. However, the endothelial protection effect and the probable protein targets of CE have not been fully characterized. In the present study, a biotin-conjugated CE analogue (BCEA) was employed as a molecular probe to research and analyse its protein targets and signaling pathways. Compared with its parental compound CE, BCEA exhibited a similar protective effect against ox-LDL induced HUVEC damage. A chemical proteomic approach identified 128 proteins that related to the cell survival signaling pathways as the targets for BCEA. Meanwhile some of these cell survival signaling pathways that showed a higher P-value in KEGG pathway analysis were associated with anti-apoptotic activity. Moreover, further evaluation with flow cytometry, JC-1 staining assays and cleaved caspase-3 activity confirmed the anti-apoptotic effect of BCEA. Taken together, these results suggested that CE can improve cell viability most likely through anti-apoptotic mechanisms, and provided the basis for the further optimization of the endothelial protection compounds.

Full text of DOI:10.1039/c6ra25572h

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PAPER
The proteomic profiling of calenduloside E targets
in HUVEC: design, synthesis and application of
biotinylated probe BCEA†
Cite this: RSC Adv., 2017, 7, 6259
Yu Tian,‡ Shan Wang,‡ Hai Shang, Min Wang, Guibo Sun,* Xudong Xu*
and Xiaobo Sun*
We previously found the total saponins from the medicinal herb Aralia elata, which exhibited strong anti-
oxidative and anti-apoptotic effects. calenduloside E (CE) is one of the major triterpenoid saponin
compounds isolated from these total saponins. However, the endothelial protection effect and the
probable protein targets of CE have not been fully characterized. In the present study, a biotin-
conjugated CE analogue (BCEA) was employed as a molecular probe to research and analyse its protein
targets and signaling pathways. Compared with its parental compound CE, BCEA exhibited a similar
protective effect against ox-LDL induced HUVEC damage. A chemical proteomic approach identified 128
proteins that related to the cell survival signaling pathways as the targets for BCEA. Meanwhile some of
these cell survival signaling pathways that showed a higher P-value in KEGG pathway analysis were
associated with anti-apoptotic activity. Moreover, further evaluation with flow cytometry, JC-1 staining
assays and cleaved caspase-3 activity confirmed the anti-apoptotic effect of BCEA. Taken together,
these results suggested that CE can improve cell viability most likely through anti-apoptotic
mechanisms, and provided the basis for the further optimization of the endothelial protection compounds.
Received 20th October 2016
Accepted 7th December 2016
DOI: 10.1039/c6ra25572h
www.rsc.org/advances
8,9
spectrometry. Furthermore, the functional validation and
pathway analysis of target proteins should also be carried out by
Introduction
Molecular target identication is one of the most signicant correlating binding site information with proteomics data for
facets and major challenges for drug discovery and develop- the sake of understanding the biological mechanism of active
10,11
ment. Although various pharmacological strategies have been compounds.
developed to screen active compounds, there are still no direct
Bioactive natural products from medicinal herbs have played
effective methods for understanding the biological action a major role in discovering novel drugs and formed a treasury
mechanism of active small molecules due to their unclear that supplies candidate drugs with structure–function diversity.
1–3
targets. In recent years, increasing studies have demonstrated Aralia elata (Miq) Seem (AS), a kind of homologous plant food
that chemical proteomics is one of the most powerful methods medicine, has been used as a tonic, antiarrhythmic, anti-arthritic,
4
for identifying the targets of small molecules. Common antihypertensive and anti-diabetic agent in traditional Chinese
12,13
approaches to identify small molecule targets involve tethering medicine.
The total saponins extracted from AS, which are
of the bioactive compounds with suitable functional groups (e.g. found to be the main pharmacological active ingredients of AS,
biotin) which serve as an affinity tag allowing the bound have been proved to exhibit anti-myocardial ischemic, anti-
5
–7
proteins to be pulled-down and isolated with streptavidin.
The isolated proteins are then separated and identied by mass and anti-apoptotic capacity.
of A. elata Xinmaitong capsules (Clinical Trial Approval Number
003L01111 by China Food and Drug Administration), AS was
developed for the treatment of coronary heart disease, and have
hypoxic activities, anti-oxidative as well as anti-inammatory
14–16
Moreover, as main components
2
Beijing Key Laboratory of Innovative Drug Discovery of Traditional Chinese Medicine
Natural Medicine) and Translational Medicine, Key Laboratory of efficacy evaluation
(
17
been successfully completed Phase III clinical trials in China.
of Chinese Medicine against glyeolipid metabolism disorder disease, State
Administration of Traditional Chinese Medicine, Key Laboratory of Bioactive
Substances and Resource Utilization of Chinese Herbal Medicine, Ministry of identied from AS.
Education, Institute of Medicinal Plant Development, Chinese Academy of Medical
Various oleanane-type triterpene saponins were extracted and
13,18
A previous report demonstrated that some
triterpenoid saponin analogue from AS could tremendously
protect the endothelial cells from injury and reduce apoptotic
Sciences, Peking Union Medical College, No. 151, Malianwa North Road, Haidian
District, Beijing 100193, P. R. China. E-mail: ytian@implad.ac.cn; sunguibo@126.com
19–21
endotheliocytes.
calenduloside E (CE, Fig. 1A) is one of the
†
1
‡
Electronic supplementary information (ESI) available. See DOI:
0.1039/c6ra25572h
major natural pentacyclic triterpenoid saponins isolated from
These two authors contributed equally to this paper.
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Fig. 1 The design of biotinylated probe BCEA from lead compound
calenduloside E (CE). (A) The chemical structure of CE. (B) The
construction of biotinylated probe BCEA. The saponin scaffold of CE
was used as the active moiety. The PEG chain coupled 1,2,3-triazole
which synthesized by a Cu(I) catalyzed Huisgen 1,3-dipolar cycload-
dition reaction was employed as the linker. The biotin moiety was
applied as the tag that bound to the streptavidin resin to immobilize
and isolate the complexation of probe and target.
22–24
AS.
However, the endothelial protection effect and protein
targets analysis of CE have not been investigated.
Scheme
1
Synthesis of biotinylated probe BCEA. Reagents and
According to previous studies, the analogs of CE which conditions: (a) BnBr, K
2
CO
3
, TBAB, DCM–H
2
O, rt, 18 h; (b) glycosyl
, Pd–C (10%), EtOAc,
donor i, TMSOTf, 4 A˚ MS, DCM, rt, 2–4 h; (c) H
2
derived from amidation at the C-28 position with propargyl-
amine, glycosylation at the C-3 position with D-galactose or
change of aglycone from oleanane to ursane, all kept the similar
reflux, 4–6 h; (d) HOBt, EDCI, propargylamine, rt, 6–8 h; (e) NaOMe,
MeOH, rt, 2–3 h; (f) compound iii, CuTc, MeOH, rt, 12 h; (g) HOSu,
EDCI, DMF, 12 h; (h) amino-PEG -azide, DMF, TEA, 8 h.
3
25
activity of parental compound CE. In this current study, we
describe the design and construction of biotinylated-CE
analogue (BCEA, Fig. 1B). Potential saponin scaffold of CE
was chosen as the active moiety of probe, and the PEG chain
coupled 1,2,3-triazole part which synthesized by a Cu(I) cata-
lyzed Huisgen 1,3-dipolar cycloaddition reaction was appended
to the C-28 carboxylic of saponin scaffold and employed as the
hydrophilic linker. The biotin moiety was applied as the tag that
bound to the streptavidin resin to immobilize and isolate the
followed by deprotection of glycosyl-groups in the presence of
NaOMe/MeOH solution to gain Compound V. The above reac-
tion conditions were reported previously by our previous
25
patent. Biotinylated PEG chain iii with azide group exposed
was synthesized according to the follow general procedures. D-
Biotin reacted with N-hydroxysuccinimide (HOSu) in the 1-
ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride
26–28
complexes of probe and target.
(EDCI) condition to get compound ii, which was amidated with
Herein, we investigate whether the parent compound CE and
its probe BCEA can protect endotheliocytes subjected to ox-LDL
amino-PEG -azide to obtain biotinylated azides chain iii in 49%
3
total yield. In the nal step, Huisgen [3 + 2] cycloaddition of
compound V with biotinylated azides chain iii in the presence of
copper(I) thiophene-2-carboxylate (CuTC) catalyst in methanol
solution resulted in the formation of 1,4 substituted triazolyl
29
injury on human umbilical vein endothelial cells (HUVEC).
BCEA was employed as small molecular probe for the proteomic
proling of the protein targets of CE. By the approach of
chemical proteomics through liquid chromatography with
tandem mass spectrometry (LC-MS/MS), the prole of the pull-
down proteins bound to BCEA were analysed. Through func-
tional validation and pathway analysis, our data reveal that CE
may function by means of reducing apoptosis to increase
endothelial cells survival.
31,32
saponin derivatives VI (BCEA).
In vitro protective activity of CE and biotinylated probe BCEA
ox-LDL induces dysfunction and injury in vascular endothelial
cells (VECs), which are the pathogenetic basis for atheroscle-
rosis formation and normally used for estimation of
compounds potential. Moreover, biotin coupled with linker
must be attached to the active compound at a position that does
not affect the potential. Here, we evaluated the endothelial
protective effect of CE and its biotinylated probe (BCEA) on ox-
LDL induced HUVEC damage. As shown in Fig. 2, pretreating
the cells with CE or BCEA for 8 h followed by 24 h of ox-LDL
exposure respectively resulted in remarkable protective effect.
Although the activity is lower than that of the original
compound CE (1, 2 mM), the probe BECA still showed acceptable
protective activity on HUVEC (4.5, 9 mM).
Results and discussion
Chemistry
As illustrated in Scheme 1, naturally abundant ursolic acid was
treated with benzyl bromide (BnBr), potassium carbonate
solution (K CO ), and tetrabutylammonium bromide (TBAB) in
2 3
dry dichloromethane (DCM) to obtain a good yield of I. Glycosyl
donor i was prepared from galactose, conditions reported
30
previously by Schmidt. Compound I reacted with glycosyl
donor in Lewis acidic conditions trimethylsilyl tri-
uoromethanesulfonate (TMSOTf) to provided compound II,
which was subjected to hydrogenation to obtain compound III
i

Labelling prole of BCEA by chemical proteomics approach
in the presence of catalytic amount of 10% Pd–C at atmospheric We hypothesized that CE bound to certain proteins to achieve its
29
pressure. Compound IV was attained via amidation with prop- protective effect on ox-LDL induced HUVEC damage. We mixed
argylamine of C-28 carboxyl group of saponin scaffold, and then BCEA probe with HUVEC cell lysate in order to allow specic
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Fig. 3 Labelling profile of BCEA by chemical proteomics. (A) Sche-
matic illustration of BCEA–protein interactions with proteomic
profiling. HUVEC cell lysate incubated with BCEA probe (18 mM) for 1 h.
(B) The binding proteins precipitation was separated by SDS-PAGE and
stained by coomassie blue staining.
Fig. 2 Evaluation of endothelial protective effect of CE and BCEA on
ox-LDL induced HUVEC damage. Cell viability of HUVEC cells incubated
with different concentrations (1, 2 mM) of CE and (4.5, 9 mM) of BCEA for
33
peptide can act as a “protein tag” in protein identication. Only
8
h. Pretreatment with CE or BCEA significantly attenuated ox-LDL-
induced cell injury. #P < 0.01 versus control group, **P < 0.01 versus ox- proteins identied with at least one unique peptide were
LDL treatment group, *P < 0.05 versus ox-LDL treatment group.
considered statistically reliable hits, which resulted in a list of 744
proteins. To shrink the numbers of lower possibility of the
potential targets, we then got rid of the protein with the score of
binding proteins to attach to the active moiety of probe. For the zero. Consequently, we have identied 587 proteins as the more
protein isolation, the BECA–protein complexes were incubated possibility targets of CE (the full list of the quantied proteins was
with streptavidin resin to immobilize the proteins. Aer extensive shown in ESI Table 1†). Through the KEGG (Kyoto Encyclopedia
washing with binding buffer, the nonbinding proteins were of Genes and Genomes) pathway analysis of these proteins, 128
9
eluted and then the resin was boiled with SDS buffer. The main proteins involved in cell survival pathways were found including
procedure is described in Fig. 3A. We next examined the binding protein processing in endoplasmic reticulum, citrate cycle (TCA
proteins using sodium dodecyl sulfate-polyacrylamide gel elec- cycle), adherens junction, gap junction and estrogen signaling
trophoresis (SDS-PAGE) and stained the gel with coomassie blue pathway that associated with anti-apoptosis effect (Fig. 4).
staining reagent (Fig. 3B). BCEA binding proteins were separated Meanwhile, we also found some else cell survival signaling path-
by SDS-PAGE and collected by cutting the gel with the visible ways (PI3K-AKT, VEGF, MAPK and ER stress) that contributed to
protein bands. Lane 1 is the marker, lane 2 is the whole cell antiapoptotic activity. Taken together, these results suggested that
lysate. The streptavidin resin beads were washed with binding CE could greatly improve the cell viability most likely through
buffer 5 times for eliminating the nonbinding proteins. The last anti-apoptosis effect. However, it is too early to speculate which
wash elution solution was kept. Lane 3 is the last time elution protein is the role player at this stage. We will identify all the
solution. The resin beads were then denatured in SDS sample highly scored proteins on the list in our future study.
buffer. Lane 4 is the resin boiled with SDS buffer. There are two
clear visible protein band a and band b in lane 4 which can be
identied on the gel. Band a has a molecular weight between 55–
72 kDa, and band b is between 43–55 kDa.
Identication of CE targets by LC-MS/MS and pathway
analysis of CE targets
Our previous studies conrmed that some triterpenoid saponins
isolated from AS could improve cell viability by means of reducing
apoptotic cells. In order to explore the relevant mechanism of
triterpenoid saponin CE by analyzing its protein targets and
signaling pathways, the chemical proteomics has been employed
to identify the probable binding targets of CE by determining the
peptides molecular weight of digested protein. In our study,
a total of 750 proteins were successfully identied from band
10,11
a and band b using LC-MS/MS in our experiment.
A unique Fig. 4 The proteins function and pathway analysis of CE targets.
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PI(ꢀ) staining kit was obtained from Invitrogen (Eugene, OR,
USA). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] was the products of Sigma Chemical Co. (St. Louis,
MO, USA). The uorescent dye JC-1 was purchased from Sigma-
Aldrich (St. Louis, MO, USA). Caspase-3 uorometric assay kits
were acquired from BioVision (CA, USA). Analytical TLC was
performed using silica gel HF254. Preparative column chro-
1
13
matography was performed with silica gel H. H and C NMR
spectra were recorded on a Bruker Advance III 600 MHz spec-
trometer. HRMS were obtained on a Thermosher LTQ-
Orbitrap XL.
The synthesis of compounds I–V and the glycosyl donor i
were described in previous studies (Scheme 1).
Fig. 5 BCEA protected against ox-LDL-induced HUVEC apoptosis. (A)
Inhibitory effects of BCEA (9 mM, 8 h) on ox-LDL-induced HUVEC Chemistry
apoptosis were detected by using flow cytometry; (B) the protect
Synthesis of compound ii. To a solution of D-biotin (500 mg,
.05 mmol) in dry DMF (10 mL), HOSu (283 mg, 2.46 mmol) and
effect of BCEA (9 mM, 8 h) on mitochondrial membrane potential were
detected by JC-1 staining. (C) Analysis of cleaved caspase-3 activity of
BCEA (9 mM, 8 h) by spectrophotometer.
2
EDCI (470 mg, 2.46 mmol) were added and the reaction mixture
was stirred at room temperature for 12 h. Reaction was moni-
tored by TLC and the suspension was ltered out and the ltrate
was washed respectively by water and then methanol to give
BCEA protected against ox-LDL-induced HUVEC apoptosis
1
pure compound ii as white solid. (476 mg, 68% yield); H-NMR
Phosphatidylserine (PS) exposure on the extra-cellular side of
the cell membrane occurs during the early stage of apoptosis. PS
exposure was investigated through ow cytometry under
Annexin V–PI double staining. Aer the stimulation of ox-LDL
for 24 h, the percentage of Annexin V(+)/PI(ꢀ) cells increased.
In this study, the percent of Annexin V(+)/PI(ꢀ) cells was
signicantly reduced in the BCEA pretreatment group in Fig. 5A.
Molecular function and cell component analysis found
numerous mitochondrial proteins, this suggested CE may play
an important role in mitochondria. The disruption of mito-
chondrial membrane potential is an early event in the apoptotic
cascade. Mitochondrial depolarization was detected by JC-1
staining in Fig. 5B. ox-LDL group exhibited an increase in
green uorescence intensity indicating mitochondrial dissipa-
tion, while the BCEA treatment group showed clearly red uo-
rescence, indicating that BCEA can protect the cells against ox-
LDL injury. Caspase-3 activity is the important part during
apoptosis, we assessed caspase-3 activation using a uorogenic
(
600 MHz, DMSO-d
6
) d: 6.40 (s, 1H, –CONH–CH–), 6.34 (s, 1H,
CONH–CH–), 4.32–4.30 (m, 1H, –CONH–CH–), 4.16–4.14 (m,
H, –CONH–CH–), 3.12–3.09 (m, 1H, –CH–S–), 2.85–2.81 (m,
H, –CO–CH –CH –CO–, –CH
–S–), 2.67 (t, J ¼ 7.5 Hz, 2H, –CO–
–), 2.58 (d, J ¼ 12.4 Hz, 1H, –CH –S–), 1.67–1.39 (m, 6H,
–CH–); C-NMR (150 MHz, DMSO-d ) d: 170.2,
68.9, 162.6, 61.0, 59.2, 55.2, 39.9, 30.0, 27.8, 27.5, 25.4, 24.3.
Synthesis of compound iii. To a solution of compound ii
100 mg, 0.29 mmol) in DMF (1 mL) was dropped 11-azido-3,6,9-
trioxaundecan-1-amine (63.3 mg, 0.29 mmol, 58 mmL) and TEA
58.7 mg, 0.58 mmol, 80 mL). The mixture was reacted at room
–
1
5
2
2
2
CH
CH
2
2
1
3
–
2
–CH
2
–CH
2
6
1
(
(
temperature for 8 h. When complete, the solvent was evapo-
rated and the crude product was subjected to column chroma-
tography (eluent: CH Cl –CH OH, 15 : 1) to gain pure
2 2 3
compound iii as white solid. (92.8 mg, 72% yield); H-NMR (600
MHz, MeOD) d: 4.51–4.48 (m, 1H, –CONH–CH–), 4.32–4.30 (m,
1
1
3
CH
1
H, –CONH–CH–), 3.68–3.61 (m, 10H, –(OCH
.56–3.54 (m, 2H, –OCH –CH –N ), 3.39–3.35 (m, 4H, –CONH–
–, –COCH
–), 3.23–3.19 (m, 1H, –CH–S–), 2.93 (dd, J ¼
2.8 Hz, 5.0 Hz, 1H, –CH –S–
–S–), 2.71 (d, J ¼ 12.8 Hz, 1H, –CH
, 2.24–2.21 (m, 2H, –OCH –CH –N ), 1.77–1.57 (m, 4H, –CH
CH –CH –CH–), 1.48–1.41 (m, 2H, –CH
NMR (150 MHz, MeOD) d: 176.1, 166.0, 71.7, 71.6, 71.5, 71.3,
1.1, 70.6, 63.4, 61.6, 56.9, 51.8, 41.0, 40.4, 36.8, 29.7, 29.5, 26.8;
2 2 2 2
CH ) –OCH –),
2
2
3
substrate
named
Asp–Glu–Val–Asp–7-amino-4
tri-
2
2

uoromethylcoumarin (DEVD-AFC). As shown in Fig. 5C,
2
2
pretreatment of BECA signicantly inhibited active caspase-3
levels, which were increased in response to ox-LDL treatment.
)
2
2
3
2
–
1
3
2
2
2 2 2
–CH –CH –CH–); C-
7
Experimental
+
32 6 5
HRMS calcd mass for C18H N O SNa [M + Na] 467.2053,
All the reagents were used without further purication unless found 467.2051.
otherwise specied. Solvents were dried and redistilled prior to Synthesis of compound VI (BCEA). To a solution of
use in the usual manner. Human umbilical vein endothelial compound V (10.0 mg, 15.2 mmol) in methanol (1 mL), CuTc
cells (HUVECs) were isolated from several fresh human umbil- (0.29 mg, 1.52 mmol) were added at room temperature. To this
ical veins using 0.1% collagenase I. The neonate cords were mixture, compound iii (6.8 mg, 15.2 mmol) was added and the
donated by the Maternal and Child Care Service Centre in Bei- reaction mixture was stirred until its completion. The methanol
jing, China. Human ox-LDL (by copper ion-induced LDL was evaporated and the crude product was puried through
ꢀ
1
oxidation, malondialdehyde >25 nmol mg ) were obtained column chromatography (eluent: CH
2 2 3 2
Cl –CH OH–H O,
from Union-Bio Technology (Beijing, China). Annexin V(+)/ 50 : 10 : 1) to offer pure compound V as white solid. (13.5 mg,
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1
0
8
8
1% yield). H-NMR (600 MHz, pyridine-d ) d: 8.54 (s, 1H, N -H), disulde bonds of proteins and then the resulting sulydryl
5
0
0
.14 (s, 1H, H-33), 8.07 (t, J ¼ 5.3 Hz, 1H, N-H), 7.48 (s, 2H, N - functional groups were alkylated by 100 mM iodoacetamide
0
H), 5.48 (t, J ¼ 3.4 Hz, 1H, H-12), 4.89 (d, J ¼ 7.7 Hz, 1H, H-1 ), (IAA, Amersham) in 25 mM NH HCO at room temperature in
4
3
4
.82 (d, J ¼ 5.3 Hz, 2H, H-31), 4.63–4.61 (m, 3H), 4.54–4.47 (4H, the dark for 0.5 h. Subsequently, the proteins were digested with
ꢀ1
m), 4.37–4.35 (1H, m, H-49), 4.19 (dd, J ¼ 3.4 Hz, 9.5 Hz, 1H), 20 ng mL porcine trypsin (modied proteomics grade, Sigma)
ꢂ
4
(
.14 (t, J ¼ 6.0 Hz, 1H), 3.87 (t, J ¼ 5.1 Hz, 2H, H-35), 3.70–3.68 overnight at 37 C. The resulting tryptic peptides mixture was
4H, m, H-36, 37), 3.63–3.58 (8H, m, H-38, 39, 40, 41), 3.42 (dd, J extracted twice from the gel pieces with 5% triuoroacetic acid
¼
11.8 Hz, 4.4 Hz, 1H, H-3), 3.22–3.19 (m, 1H, H-47), 2.93 (dd, J (TFA, Fluka) in 50% ACN solution. The pooled extracts were
12.5 Hz, 4.9 Hz, 1H, H-48-1), 2.87 (d, J ¼ 12.5 Hz, 1H, H-48-2), evaporated in a vacuum centrifuge (Labconco, Kansas, MO),
¼
2
.42 (d, J ¼ 10.7 Hz, 1H, H-18), 2.38 (t, J ¼ 7.4 Hz, 2H, H-43), 1.33 and resuspended with 0.1% methanoic acid (Sigma) prior to the
(s, 3H, CH ), 1.23 (s, 3H, CH ), 1.00 (s, 3H, CH ), 0.97 (d, J ¼ LC separation and MS detection.
3
3
3
6
.4 Hz, 3H, CH ), 0.95 (s, 3H, CH ), 0.91 (s, 3H, CH ), 0.80 (s, 3H,
LC-MS/MS analysis. All digested peptide mixtures were
) d: 177.6, 173.0, 164.3, separated by reverse phase (RP) HPLC followed by tandem MS
3
3
3
1
3
CH
3
); C-NMR (150 MHz, pyridine-d
5
1
7
4
3
2
45.6, 139.2, 126.1, 123.8, 107.5, 88.8, 76.7, 75.4, 73.1, 70.7, 70.6, analysis. RP-HPLC was performed on a surveyor LC system
0.6, 70.5, 70.4, 70.3, 69.7, 62.4, 62.3, 60.5, 56.2, 55.8, 53.5, 50.2, (Thermo Finnigan, San Jose, CA). Samples were loaded into
7.9, 47.7, 42.5, 41.0, 39.9, 39.8, 39.7, 39.5, 39.3, 38.9, 37.9, 36.8, a trap column (Zorbax 300SB-C18 peptide traps, 300 mm ꢁ 65
6.1, 35.6, 33.3, 31.1, 29.0, 28.9, 28.3, 28.2, 26.7, 26.0, 24.9, 23.8, mm, Agilent Technologies, Wilmington, DE) rst at a 3 mL
ꢀ
1
3.6, 21.3, 18.4, 17.4, 17.0, 16.9, 15.7; HRMS calcd mass for min ow rate aer the split for peptides enrichment and
+
ꢀ1
C H N O SNa [M + Na] 1122.6501, found 1122.6490.
desalting. Aer ow-splitting down to about 1.5 mL min ,
5
7
93 7 12
peptides were transferred to the analytical column (RP-C18, 150
mm ꢁ 150 mm, Column Technology, Inc., Fremont, CA) for
separation with a 195 min linear gradient from 96% buffer A
Biological studies
Cell viability analysis. Cell viability was determined by the 3- (0.1% methanoic acid in water) to 50% buffer B (84% ACN, 0.1%
ꢀ
1
(
4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) methanoic acid in water) at a ow rate of 250 nL min . The
22
assay as previously described. Briey, HUVEC cells were plated analytical column was regenerated for 20 min with buffer A
4
on 96-well plates at a density of 8 ꢁ 10 cells per well. The cultures before loading the next sample. A thermo QE linear ion trap
ꢂ
were grown at 37 C for 24 h. Then the cells were pretreated mass spectrometer equipped with a nanospray source was used
respectively with CE (1, 2 mM) or BCEA (4.5, 9 mM) for 8 h, then for the MS/MS experiment in the positive ion mode. The
ꢀ
1
ꢀ1
ꢂ

owed by ox-LDL (70 mg mL , 24 h). 20 mL MTT (5 mg mL ) was temperature of the ion transfer capillary was set at 160 C. The
added to each well and incubated for 4 h. The medium was then spray voltage was 3.0 kV and normalized collision energy was set
removed, and the formazan crystals were dissolved with dimethyl at 35.0% for MS/MS. The MS analysis was performed with one
sulphoxide (DMSO). Absorbance was read at 570 nm on a micro- full MS scan (m/z 400–1800) followed by 10 MS/MS scans on the
plate reader (TECAN Innite M1000, Austria).
10 most intense ions from the MS spectrum with the dynamic
7
Biotin–neutravidin pull-down assay. HUVEC cells (1.0 ꢁ 10 ) exclusion settings: repeat count 2, repeat duration 30 s, exclu-
were disrupted in RIPA lysis buffer and sonicated with freshly sion duration 90 s. Data were acquired in data-dependent mode
added protease inhibitor cocktail (CST). The cell lysate (200 mL) using Xcalibur soware.
was incubated with 2 mL BCEA (18 mM) at room temperature for
Bioinformatics analysis. Tandem mass spectra were
1
h. The mixture was further incubated with equilibrated and searched against mascot 2.1 (Local Host) RAT protein database.
packed streptavidin resin at room temperature for 1 h, which The search results were then ltered using a cut off of 1% for
was followed by centrifugation (1 min, 2500 rpm) and ve times peptide false identication rate. Peptides with Z score < 4 or
repeated washing with 0.5 mL binding buffer to wash out delta-mass > 5 ppm were rejected. Furthermore, the minimum
nonbinding proteins. The pull-down assay was performed number of peptides to identify a protein was set to 1. The
according to the protocol of streptavidin (20347 Pierce® Strep- default parameters for the Quantitative soware Prole Analysis
tavidin Agarose Resins). The resin was boiled with 2ꢁ SDS 2.0 soware were used throughout the analysis. Bioinformatics
loading buffer for 10 min and then electrophoresed on a 10% analysis was carried out to categorize proteins based on bio-
SDS-polyacrylamide gel. The resulting gel was visualized with logical processes, cellular component and molecular function
coomassie blue staining.
using annotations in Protein ANalysis THrough Evolutionary
(PANTHER) database 6.1 (http://
In gel digestion. The CBB-stained SDS-PAGE gel lane was Relationships
v
manually cut into 6 slices depending on protein molecular www.pantherdb.org), which is in compliance with gene
weight (MW). Each slice was diced into 1 mm ꢁ 1 mm pieces, ontology (GO) standards. Signaling pathway analysis was per-
and then subjected to in-gel tryptic digestion, essentially as formed with the tools on the Kyoto Encyclopedia of Genes and
34
described by Wilm. Briey, the gel pieces were rinsed thrice Genome (KEGG) database (http://www.genome.jp/kegg/
using Milli-Q water (Millipore) and destained twice with 25 mM pathway.html), respectively. Identied proteins in different
ꢂ
NH HCO in 50% acetonitrile (ACN, Amer-sham) at 37 C until groups were analyzed for molecular function, biological process
4
3
the color depigmented completely. The dried gels were incu- or pathway term in PANTHER using the binomial test.
bated with 50 mM tris(2-carboxyethyl)phosphine (TCEP, Sigma)
Assessments of cell apoptosis. The HUVECs were incubated
ꢂ
ꢀ1
in 25 mM NH
4
HCO
3
at 56 C for 1 h to reductively cleave the with ox-LDL (70 mg mL , 24 h) and pretreated with BCEA (9 mM)
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h before the apoptosis analysis. For the detection of early
apoptosis and necrosis, double uorescence staining was per-
formed using an Annexin V-FITC/PI apoptosis staining kit
according to the manufacturer's instructions. Cellular uores-
cence was measured using ow cytometry analysis with a FACS
Calibur Flow Cytometer (BD Biosciences, USA).
Paper
8
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