Development of an inflammation-specific MRM assay for the

Development of an inflammation-specific MRM assay for the

Development of an inflammation-specific MRM assay for the
discovery of novel vascular pharmaceuticals from escin derivatives
Dominik Domanski, Anna Perzanowska, Michał Dadlez. Mass Spectrometry Laboratory, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw,
Poland
Katarzyna Koziak, Oliwia Zegrocka-Stendel, Dorota Maciejko, Magdalena Kowalewska. Department of Immunology, Biochemistry and Nutrition. Medical University of
Warsaw, Poland.
Introduction
Proteins significantly altered in abundance
due to escin (q-value <0.05) under the
iTRAQ: Number of proteins identified with inflammatory condition.
FDR <0.01 and 2 or more peptides in the three Accession
number q-value Ratio Peptides
Protein name
Q9Y287
0.0001 3.79
2
Integral membrane protein 2B GN=ITM2B
independent biological replicate analyses.
Neuroblast differentiation-associated protein AHNAK
Results & Discussion
Escin is a mixture of triterpene saponins isolated from seeds of the horse chestnut tree
Aesculus hippocastanum. It mainly consists of four substances that make up around 70% of
this mixture: escin Ia, escin Ib, escin IIa and escin IIb1. The extract has been extensively used
by herbal medicine in the treatment of vascular disorders due to its anti-edematous, antiinflammatory and venotonic properties2, and is an active ingredient of popular drug
formulations such as Escin, Reparil and Venitan. Despite this, its pharmacological mechanism
of action remains largely unknown. We therefore aimed to study the mechanism of escin
action on vascular endothelial cells and to identify possible restrictions on its use. Using
proteomic approaches we have determined changes in the expression level of cellular
proteins resulting from the response of human umbilical vein endothelial cells (HUVEC) to
escin. Global discovery-type proteomic methods (isobaric tags for relative and absolute
quantitation: iTRAQ) were used on HUVEC cells under inflammatory conditions induced by
tumor necrosis factor (TNF), and targeted proteomic methods (multiple reaction monitoring:
MRM) were used to verify the results and create an inflammation-specific MRM panel of
proteins that revealed escin’s actions under inflammatory and non-inflammatory conditions.
Discovery global analysis – iTRAQ
HUVEC +/- TNF + escin
Protein markers mechanistic information
Targeted analysis - MRM
Proteins significantly altered in abundance due
to TNF (q-value <0.05).
Accession
number q-value
P05121
0.0003
Q9H4M9 0.0003
P19320
0.0003
P31689
0.0003
P04275
0.0003
P16581
0.0003
P07996
0.0003
P07942
0.0003
P04179
0.0003
P05362
0.0003
Q9P2E9
0.0011
Q13201
0.0015
P06756
0.0057
P11940
0.0198
Q9Y4L1
0.0317
Q96QV1 0.0318
Q9Y376
0.0367
Ratio
2.4
1.23
4.82
1.33
0.7
7.85
1.12
0.87
3.29
3.71
1.1
0.82
1.14
1.11
1.1
0.88
1.16
Peptides
18
14
23
10
85
14
38
15
8
17
59
17
18
19
43
11
8
Protein name
Plasminogen activator inhibitor 1 GN=SERPINE1
EH domain-containing protein 1 GN=EHD1
Vascular cell adhesion protein 1 GN=VCAM1
DnaJ homolog subfamily A member 1 GN=DNAJA1
von Willebrand factor GN=VWF
E-selectin GN=SELE
Thrombospondin-1 GN=THBS1
Laminin subunit beta-1 GN=LAMB1
Superoxide dismutase [Mn], mitochondrial GN=SOD2
Intercellular adhesion molecule 1 GN=ICAM1
Ribosome-binding protein 1 GN=RRBP1
Multimerin-1 GN=MMRN1
Integrin alpha-V GN=ITGAV
Polyadenylate-binding protein 1 GN=PABPC1
Hypoxia up-regulated protein 1 GN=HYOU1
Hedgehog-interacting protein GN=HHIP
Calcium-binding protein 39 GN=CAB39
HUVEC +/- TNF +/- escin
Main compounds within the escin extract: escin Ia: R1=T, R2=-COCH3,
R3=-H, escin Ib: R1=A, R2=-COCH3, R3=-H, escin IIa: R1=T, R2=-H, R3=COCH3,, escin IIb: R1=A, R2=-H, R3=-COCH3.
Verification & MRM Panel
for future Rx selection
Materials & Methods
For the iTRAQ analysis HUVEC cells were pre-incubated with escin (3 mM) and then treated
with TNF and escin for the last 6 hours. All biological replicates were from different donors (9
in total), mixed into three groups to reduce the effect from individual variability.
For the MRM
analysis HUVEC cells
from 5 individual
donors were
analyzed
independently. The
effect of escin was
tested at two
different
concentrations (2 & 3
mM) and also under
non-inflammatory
conditions.
STRING analysis of escin
action – iTRAQ results
18 IEF
fractions
3 h. nano-LC-MS/MS
on LTQ-Orbitrap
Velos
0.120
0.100
VCAM1
*
MRM: Control / TNF / Escin (2 & 3 mM) / Escin (2 & 3 mM) +TNF
Protein name
Peptide sequence
Vascular cell adhesion protein 1 (VCAM1)
SLEVTFTPVIEDIGK
MRM panel
Plasminogen activator inhibitor 1
optimization: 24
von Willebrand factor
proteins, 48 stableisotope-labeled
E-selectin
standard (SIS)
Intercellular adhesion molecule 1 (ICAM1)
peptides (13C,15NSuperoxide dismutase [Mn], mitochondrial
K/R)
DnaJ homolog subfamily A member 1
Protein name
COUP transcription factor 2
GIQVELYSFPR
SLTLDVQGR
Tumor necrosis factor alpha-induced protein 2
GAVDQLTR
VFQQVAQASK
**
0.060
0.040
0.020
0.000
CD63 antigen
Hedgehog-interacting protein
LLDLVFLLDGSSR
NWAPGEPNNR
Low affinity cationic amino acid transporter 2
YTHLVAIQNK
ASVSVTAEDEGTQR
Thrombospondin-1
VELAPLPSWQPVGK
Multimerin-1
Endothelial protein C receptor
0.400
VMSEFNNNFR
0.350
VFLEVAELHR
0.300
EPYLDIHK
0.250
SPLPVAFEYVGWGPAK
0.200
NLSSPFIFHEK
0.150
GGVNDNFQGVLQNVR
FVFGTTPEDILR
LPASFDAR
AIWNVINWENVTER
Palmitoyl-protein thioesterase 1
ETIPLQETSLYTQDR
GDVTAQIALQPALK
*
* **
**
Amyloid beta A4 protein
THPHFVIPYR
LALENYITALQAVPPRPR
Matrix metalloproteinase-14
GLPASINTAYER
HWVFDEASLEPGYPK
ELVNNLGEIYQK
Podocalyxin
LASVPGSQTVVVK
HSLPDIQLLQK
Cathepsin Z
VGDYGSLSGR
GLTEFVEPIIQIK
Tyrosine-protein kinase JAK1
TSFQNLIEGFEALLK
TLAFPLTIR
Integral membrane protein 2B
IENIDHLGFFIYR
NLLELLINIK
References
1.
2.
3.
The project „Development of targeted MS-based proteomic assays for the discovery of a novel endothelium-specific pharmaceutical for the treatment of human vascular
diseases" was carried out as HOMING PLUS Program of Foundation for Polish Science. The project is co-financed from the European Union - Regional Development Fund.
Chen J et al., (2007) Determination of four major saponins in the seeds of Aesculus chinensis Bunge using accelerated solvent extraction followed by HPLC
and ESI-TOF-MS. Anal Chim Acta. 2007 Jul 23;596(2):273-80.
Sirtori, C. R. (2001). "Aescin: pharmacology, pharmacokinetics and therapeutic profile." Pharmacol Res 44(3): 183-193.
Franceschini A et al., (2013) STRING v9.1: protein-protein interaction networks, with increased coverage and integration. Nucleic Acids Res. Jan:41.
0.050
0.040
**
**
0.9
1.56
0.86
1.18
1.21
0.64
0.69
1.25
1.3
2
0.91
1.42
0.62
1.2
1.1
204
6
28
17
53
5
23
11
17
7
115
14
9
19
16
O00469
Q9H223
0.0001
0.0003
0.81
1.11
17
19
GN=AHNAK
Podocalyxin GN=PODXL
Vigilin GN=HDLBP
Integrin beta-1 GN=ITGB1
A-kinase anchor protein 12 GN=AKAP12
Cathepsin B GN=CTSB
Vascular cell adhesion protein 1 GN=VCAM1
Hedgehog-interacting protein GN=HHIP
Purine nucleoside phosphorylase GN=PNP
Amyloid beta A4 protein GN=APP
Filamin-A GN=FLNA
E-selectin GN=SELE
Palmitoyl-protein thioesterase 1 GN=PPT1
Cell surface glycoprotein MUC18 GN=MCAM
Glutathione S-transferase omega-1 GN=GSTO1
Procollagen-lysine,2-oxoglutarate 5-dioxygenase 2
GN=PLOD2
EH domain-containing protein 4 GN=EHD4
P27105
P29279
O75923
O94832
P08648
Q96HC4
Q14392
P23458
Q9UNN8
Q01995
Q15392
P09493
P05783
P17813
Q9NPY3
Q13907
Q9NY15
P35241
P07951
P51813
P42892
P00352
P14324
Q13201
Q92503
P08729
0.0003
0.0004
0.0005
0.0005
0.0005
0.0005
0.0005
0.0005
0.0008
0.001
0.0011
0.0015
0.0018
0.0039
0.0044
0.0045
0.0047
0.0053
0.006
0.0064
0.0064
0.0078
0.0082
0.0096
0.0096
0.0097
1.16
0.73
1.22
1.15
1.18
0.89
1.19
1.32
0.61
0.75
1.33
0.78
0.88
1.21
1.31
1.3
0.69
1.26
0.69
0.85
1.16
0.88
1.22
0.83
1.39
0.79
7
6
18
20
19
19
3
8
4
11
6
5
12
15
7
7
9
13
4
12
18
19
10
17
5
11
Erythrocyte band 7 integral membrane protein GN=STOM
Connective tissue growth factor GN=CTGF
Dysferlin GN=DYSF
Myosin-Id GN=MYO1D
Integrin alpha-5 GN=ITGA5
PDZ and LIM domain protein 5 GN=PDLIM5
Leucine-rich repeat-containing protein 32 GN=LRRC32
Tyrosine-protein kinase JAK1 GN=JAK1
Endothelial protein C receptor GN=PROCR
Transgelin GN=TAGLN
Delta(24)-sterol reductase GN=DHCR24
Tropomyosin alpha-1 chain GN=TPM1
Keratin, type I cytoskeletal 18 GN=KRT18
Endoglin GN=ENG
Complement component C1q receptor GN=CD93
Isopentenyl-diphosphate Delta-isomerase 1 GN=IDI1
Stabilin-1 GN=STAB1
Radixin GN=RDX
Tropomyosin beta chain GN=TPM2
Cytoplasmic tyrosine-protein kinase BMX GN=BMX
Endothelin-converting enzyme 1 GN=ECE1
Retinal dehydrogenase 1 GN=ALDH1A1
Farnesyl pyrophosphate synthase GN=FDPS
Multimerin-1 GN=MMRN1
SEC14-like protein 1 GN=SEC14L1
Keratin, type II cytoskeletal 7 GN=KRT7
P16284
0.0105
1.16
21
Q01581
P17301
Q9BXS4
P14923
P17302
O00170
P37268
0.012
0.0123
0.0124
0.0125
0.0151
0.0153
0.0153
1.13
1.1
2.6
1.18
1.58
0.81
1.36
12
17
2
12
2
6
9
P11717
0.0153
1.41
5
P23467
Q05682
Q9UBR2
P48449
Q14254
P49588
P68871
P02042
P08962
P48509
Q9BWD1
Q16222
Q13310
Q9UDY4
P10619
0.0165
0.0184
0.0198
0.0204
0.0208
0.0218
0.0229
1.32
0.88
0.56
1.2
0.88
0.87
2.1
10
18
4
6
9
27
2
0.0293
0.0323
0.0375
0.0377
0.042
0.0422
0.0446
1.85
1.34
1.19
0.82
0.9
0.9
0.74
2
3
8
7
13
13
5
Q99613
0.0478
0.9
22
Platelet endothelial cell adhesion molecule GN =PECAM1
Hydroxymethylglutaryl-CoA synthase, cytoplasmic
GN=HMGCS1
Integrin alpha-2 GN=ITGA2
Transmembrane protein 59 GN=TMEM59
Junction plakoglobin GN=JUP
Gap junction alpha-1 protein GN=GJA1
AH receptor-interacting protein GN=AIP
Squalene synthase GN=FDFT1
Cation-independent mannose-6-phosphate receptor
GN=IGF2R
Receptor-type tyrosine-protein phosphatase beta
GN=PTPRB
Caldesmon GN=CALD1
Cathepsin Z GN=CTSZ
Lanosterol synthase GN=LSS
Flotillin-2 GN=FLOT2
Alanine--tRNA ligase, cytoplasmic GN=AARS
Hemoglobin subunit beta GN=HBB
Hemoglobin subunit delta GN=HBD
CD63 antigen GN=CD63
CD151 antigen GN=CD151
Acetyl-CoA acetyltransferase, cytosolic GN=ACAT2
UDP-N-acetylhexosamine pyrophosphorylase GN=UAP1
Polyadenylate-binding protein 4 GN=PABPC4
DnaJ homolog subfamily B member 4 GN=DNAJB4
Lysosomal protective protein GN=CTSA
Eukaryotic translation initiation factor 3 subunit C
GN=EIF3C
0.000
PODXL
*
* **
0.500
0.000
CTSZ
0.120
0.100
0.080
* **
* **
0.060
PPT1
1.000
0.900
0.800
0.700
0.600
0.500
0.400
0.300
0.200
0.100
0.000
0.250
* **
* **
0.100
0.050
0.000
0.000
0.000
* **
MMP14
*
* **
**
**
0.250
0.200
CD63
*
** * **
0.150
0.100
0.050
0.000
VMSEFNNNFR
PROCR
*
0.150
0.020
EHD1
**
0.200
0.040
*
SERPINE1
*
VFQQVAQASK
GLPASINTAYER
0.050
0.090
0.080
0.070
0.060
0.050
0.040
0.030
0.020
0.010
0.000
0.200
0.180
0.160
0.140
0.120
0.100
0.080
0.060
0.040
0.020
0.000
GDVTAQIALQPALK
LASVPGSQTVVVK
VGDYGSLSGR
**
0.020
0.100
0.100
LPASFDAR
DNAJA1
0.040
0.200
0.050
*
0.060
0.300
0.250
**
0.100
NLSSPFIFHEK
0.600
*
0.080
0.400
0.150
0.000
**
0.000
0.200
* **
**
0.040
0.700
SOD2
0.120
0.010
* **
ITM2B
* **
0.160
0.140
0.050
NLLELLINIK
* **
*
0.020
0.000
CTSB
SLC7A2
0.030
0.010
0.100
0.450
0.400
0.350
0.300
0.250
0.200
0.150
0.100
0.050
0.000
0.070
0.060
0.020
TIVITSHPGQIVK
Fragment ion
selection &
collision energy
voltage
optimization
*
0.030
IPGIYVLSLEIGK
IINTPEVVR
TQSGLQSYLLQFHGLVR
0.060
THPHFVIPYR
NNHTASILDR
Cathepsin B
TIVITSHPGQIVK
APP
SHGFDTLLQNLHEDLKPLFK
DGTFPLPIGESVTVTR
ILEVHIDK
EH domain-containing protein 1
SQYPNQPTR
0.200
0.180
0.160
0.140
0.120
0.100
0.080
0.060
0.040
0.020
0.000
TNFAIP2
VLLVELPAFLR
VLLVELPAFLR
YTLFQIFSK
ILAGPAGDSNVVK
Peptide sequence
TVSSSVIEQLFFVR
HYNGEAYEDDEHHPR
Parent ion
selection &
Cone voltage
optimization
**
0.045
0.040
0.035
0.030
0.025
0.020
0.015
0.010
0.005
0.000
GIQVELYSFPR
Mascot & Diifprot
data analysis
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001
STRING3 gene ontology analysis of iTRAQ results reveals that escin might have an effect on cell
adhesion proteins involved in wound healing/response (red) and on cholesterol biosynthesis
proteins (red + green circle).
0.080
iTRAQ:
Control / TNF /
Escin (3 mM) +TNF
Q09666
O00592
Q00341
P05556
Q02952
P07858
P19320
Q96QV1
P00491
P05067
P21333
P16581
P50897
P43121
P78417
ETIPLQETSLYTQDR
**
**
TQSGLQSYLLQFHGLVR
MRM analysis: Changes in relative protein
concentrations for representative peptides in the
MRM panel showing effects of escin under
inflammatory and non-inflammatory conditions.
Significance: p<0.05: *=to C, **=to TNF.
ELVNNLGEIYQK
Conclusions
Escin was shown to have an inhibitory effect on cell adhesion proteins involved in the
inflammatory response as well as an inducing effect on the cholesterol biosynthesis pathway
in HUVEC cells. Under non-inflammatory conditions escin has an effect on lysosomal proteins
and the amyloidogenic pathway. The MRM panel of inflammation-specific proteins will be
useful in the selection of active escin derivatives on the endothelium in the future.