Dr Cédric DELPORTE
Prof. Ass. Dr Pierre VAN ANTWERPEN
Laboratory of Pharmaceutical Chemistry
&
Analytical Platform of the Faculty of Pharmacy (ULB)
POLARIS CHIP FOR PROTEOMIC’S:
PROTEIN AND PEPTIDE PROFILING
Agilent MS User Meeting 1st July 2014 2
Introduction
Atherosclerosis
LC-MS based proteomics in atherosclerosis:
Example of Apolipoprotein B100
Applications
Detecting oxidative post-translationnal modifications in vitro
Nano-LC Chip Cube system and POLARIS Chip
Detecting oxidative post-translationnal modifications in vivo
Complex samples
Atheromatous plaque
Conclusions and Perspectives
Overview
Agilent MS User Meeting 1st July 2014
Introduction
Atherosclerosis
Inflammatory disease
Oxidized LDLs (oxLDLs)
Myeloperoxidase (MPO)
MoxLDLs
HOCl H2O2, Cl-
3/16
Agilent MS User Meeting 1st July 2014 4
Delporte et al, Mediators of inflammation 2013
Atherosclerosis
Nat-LDL Mox-LDL Monocyte/ Macrophage
Scavenger receptor
Inti
ma
H2O2
Endothelial cell
Foam Cell
PMN Neutrophil
MPO
Primary granule
NOX2
O2•-
H2O2
HOX
SOD
AT-R1
AT II
Mox-LDL
Nat-LDL
IL-8
TNFα
LDL Oxidation • Theory says: “LDLs are oxidized in
the intima (subendothelial spaces of
arteries)”
• Experiments show also that
oxidation occurs at the surface of
endothelial cells in plasma
Introduction
Agilent MS User Meeting 1st July 2014
Example of Apolipoprotein B100
5
Protein of the low-density lipoprotein (LDLs)
Includes 4536 amino acids
Mass: ≈ 550kDa
N-Glycosylation (19 sites)
Modified ApoB100
Atherogenesis
Recovery of 50 %
in literature
Agilent MS User Meeting 1st July 2014
Example of Apolipoprotein B100
6
LDL Isolation
Protein Isolation
Protein Unfolding and Digestion -Reduction – Alkylation
- Trypsin or Glu-C digestion
-Deglycosylation by PNGase
-One step processing using RapiGest or TFE
Peptide Purification - Ion-pairing reagent
- Washing volumes
- RapiGest
- TFE
Peptide Separation - Gradient
MS/MS Analysis - Ionization - Multiple Injection
- Fragmentation - Data acquisition
Data Analysis - Database - MS/MS Search
- Validation - Data extraction
Steps:
Spectum Mill
Agilent MS User Meeting 1st July 2014
Example of Apolipoprotein B100
7
Validation of false-positive peptides
Default automatic validation + manual validation
99 % of sequence
recovery
90 % of sequence
recovery
Agilent MS User Meeting 1st July 2014 8
Introduction
Atherosclerosis
LC-MS based proteomics in atherosclerosis:
Example of Apolipoprotein B100
Applications
Detecting oxidative post-translationnal modifications in vitro
Nano-LC Chip Cube system and POLARIS Chip
Detecting oxidative post-translationnal modifications in vivo
Complex samples
Atheromatous plaque
Conclusions and Perspectives
Overview
Agilent MS User Meeting 1st July 2014
Example of Apolipoprotein B100
9
Modified peptides (PTMs)
4 fold higher
Agilent MS User Meeting 1st July 2014
Example of Apolipoprotein B100
10
LDL oxidation in vitro by MPO vs HOCl
Detection of MPO-specific oxidation:
MPOsyst 50 and 100 µM
MPOsyst 100 µM
10
Oxidation MPO
(MoxLDL)
Chemical Oxidation Enzymatic Oxidation
To mimic MPO/H2O2/Cl-
*
Agilent MS User Meeting 1st July 2014
LDL analysis of patients submitted to
hemodialysis
LDL isolation from 9 patients and 9
volunteers
ApoB100 isolation
Protein treatment (1 mg/ml)
Analysis by RRLC-QTOF ( 200 µg Eq
protein injected)
W 1108
M 723 and 1285
Very few PTMs recovered
Analysis by LC-CHIP/MS ???
11
Example of Apolipoprotein B100
Agilent MS User Meeting 1st July 2014
Example of Apolipoprotein B100, Polaris CHIP
12
Nano LC-Chip Cube MS system
Agilent MS User Meeting 1st July 2014
Example of Apolipoprotein B100, Polaris CHIP
13
Rotor
Stator
inner rotor
outer rotor
Stator
Rotor
Side View
Nanopump
Autosampler
Waste
Microvalve
HPLC-Chip
Agilent MS User Meeting 1st July 2014
Example of Apolipoprotein B100, Polaris CHIP
Combines:
Enrichment column (cleaning and concentrating sample)
Analytical column (peptide separation)
Needle (spray/ionization)
Advantages:
For low quantities and dirty samples, increasing sensitivity
Friendly-user system (needle position easy)
Disadvantages:
Need nano and capillary pumps (whether you work on RRLC)
Small section tubing that needs to be handled carefully
Agilent MS User Meeting 1st July 2014
CHIP characteristics Prot Id-CHIP 150 Polaris HR CHIP
Phase, chemistry Zorbax C18 Polaris C18
Particule size 5 µm 3 µm
Porosity 300 Å 180 Å
Enrichment column 40 nl 360 nl
Analytical column 150 x 75 µm 150 x 75 µm
Pressure 200 Bars (150) 250 Bars (200)
Volume injected 1 µl 1 µl
Capillary flow 4 µl/min 2 µl/min
Nano flow 0.2-0.6 µl/min (0.4) 0.2-0.45 µl/min (0.3)
Increase resolution
Example of Apolipoprotein B100, Polaris CHIP
Agilent MS User Meeting 1st July 2014
LDL analysis of patients submitted to
hemodialysis
LDL isolation from 9 patients and 9
volunteers
ApoB100 isolation
Protein treatment (1 mg/ml)
Analysis by RRLC-QTOF ( 200 µg Eq
protein injected) 90 % recovery
W 1108
M 723 and 1285
Very few PTMs recovered
Analysis by LC-CHIP-QTOF ( 20 µg Eq
protein injected)
Prot-Id CHIP 150 no interesting
modifications observed
Polaris CHIP 70 % recovery
90 PTMs detected including 36 new
Y 125 and M 2499
16
Example of Apolipoprotein B100, Polaris CHIP
Agilent MS User Meeting 1st July 2014 17
Introduction
Atherosclerosis
LC-MS based proteomics in atherosclerosis:
Example of Apolipoprotein B100
Applications
Detecting oxidative post-translationnal modifications in vitro
Nano-LC Chip Cube system and POLARIS Chip
Detecting oxidative post-translationnal modifications in vivo
Complex samples
Atheromatous plaque
Conclusions and Perspectives
Overview
Agilent MS User Meeting 1st July 2014
Atheromatous plaque
18
Plaques (aorta, carotids or abdominal artery) are
extracted from patients
The tissues are frozen and crushed
Agilent MS User Meeting 1st July 2014
Experimental
19/16
Extraction Buffer = 0.15 M NaCl, 100 µM DTPA 100 µM BHT, protease inhibitor mixture (50 µL/4 mL),
10 mM phosphate, pH 7.4.
≈2.5 g of powder
4 mL of Extraction buffer
Extraction 24h at 4 °C (Rotamix) (Twice)
Deplipidation of 1 mL extract
Reduction, Alkylation and Digestion by trypsin (24h)
Delipidation by 6.6 mL of
Ether/MeOH/H2O (7/3/1 v/v) (Twice)
Take ≈20 mg
Reduction, Alkylation and Digestion by
trypsin 24 h
B
A
Only delipidation (A) vs Extraction + Delipidation (B)
Agilent MS User Meeting 1st July 2014
Experimental
LC conditions
20/16
Injection Volume: 5 µL
RRLC Poroshell 120 EC-C18 2.1 x 100 mm, 2.7 µm
Gradient Time [min] % B Flow [mL/Min]
0 1 0.2
5 1 0.2
15 5 0.3
25 5 0.5
80 20 0.5
83 20 0.5
93 90 0.5
95 90 0.5
99 1 0.5
101 1 0.2
105 1 0.2
Stop Time 105
Agilent MS User Meeting 1st July 2014
Experimental
LC conditions
21/16
Injection Volume: 5 µL
Gradient Time [min] % B Gradient Time [min] % B Gradient Time [min] % B
0 1 0 1 0 1
5 1 5 1 5 1
15 5 15 5 15 5
25 5 25 5 25 5
80 20 80 20 80 20
83 20 83 20 83 20
93 90 93 90 93 90
95 90 95 90 95 90
99 1 99 1 99 1
101 1 101 1 101 1
105 1 105 1 105 1
Stop Time 105 Stop Time 105 Stop Time 105
Flow 0.60 µL/min Flow 0.40 µL/min Flow 0.40 µL/min
Sample Loading 2.0 µL at 3% B Sample Loading 2.0 µL at 3% B Sample Loading 2.0 µL at 3% B
ProtID-Chip-150 (II) 300 Å C18 0.075 x 150 mm,
40 nL trap column
Large capacity Chip (II) High Capacity Loading,
300 Å C18 0.075 x 150 mm, 160 nL trap column
Polaris-HR-Chip 3C18High Resolution Chip 150
mm 180 Å 3 µm C18, 0.075 x 150 mm, 360 nL
trap column
Agilent MS User Meeting 1st July 2014
Atheromatous plaque
22
Lots of abundant proteins
are found such as albumin,
fibrinogen, immuno-
glubulins and hemoglobin
2586
1155 1212
1577
962
552 641
922
0
500
1000
1500
2000
2500
3000
3500
RRLC Large Cap ProteinID 150 Polaris
Number of spectra
Total
witout abundant proteins
51
61 62
80
45
55 57
74
0
10
20
30
40
50
60
70
80
90
RRLC Large Cap ProteinID 150 Polaris
Total Found Proteins Total
witout abundant proteins
301
206 222
354
197 142
148
266
0
50
100
150
200
250
300
350
400
RRLC Large Cap ProteinID 150 Polaris
Number of unique peptides
Total
witout abundant proteins
Agilent MS User Meeting 1st July 2014
Atheromatous plaque
23
51
61 62
80
45
55 57
74
0
10
20
30
40
50
60
70
80
90
RRLC Large Cap ProteinID 150 Polaris
Total Found Proteins Total
witout abundant proteins
Protein Name Protein_ID
Serum albumin P02768
Serotransferrin P02787
Fibrinogen beta chain P02675
Hemoglobin subunit beta P68871
Hemoglobin subunit delta P02042
Hemoglobin subunit gamma-1 P69891
Alpha-1-antitrypsin P01009
Alpha-2-macroglobulin P01023
Actin, alpha cardiac muscle 1 P68032
Actin, cytoplasmic 2 P63261
Beta-actin-like protein 2 Q562R1
Ig gamma-2 chain C region P01859
Ig gamma-1 chain C region P01857
Ig gamma-3 chain C region P01860
Apolipoprotein A-I P02647
Haptoglobin P00738
Ig mu chain C region P01871
Complement C3 P01024
Fibrinogen gamma chain P02679
Ig alpha-1 chain C region P01876
Fibrinogen alpha chain P02671
Hemoglobin subunit alpha P69905
Lots of abundant proteins
are found such as albumin,
fibrinogen, immuno-
glubulins and hemoglobin
Agilent MS User Meeting 1st July 2014
Atheromatous plaque
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Protein Id Pept number RRLC Pept numb CHIP
Apolipoprotein A-I 10 12
Apolipoprotein A-II 0 2
Apolipoprotein A-IV 2 2
Apolipoprotein B-100 2 10
Apolipoprotein E 4 5
Total 18 31
Conclusions:
Saturation of the CHIP system with high abundance proteins
Diversity of peptides with low abundance proteins POLARIS
Bring better proteomic understanding of the plaque « Interactomics »
No pressure increase despite dirty samples
Agilent MS User Meeting 1st July 2014 25
Introduction
Atherosclerosis
LC-MS based proteomics in atherosclerosis:
Example of Apolipoprotein B100
Applications
Detecting oxidative post-translationnal modifications in vitro
Nano-LC Chip Cube system and POLARIS Chip
Detecting oxidative post-translationnal modifications in vivo
Complex samples
Atheromatous plaque
Conclusions and Perspectives
Overview
Agilent MS User Meeting 1st July 2014
Perspectives and conclusions
26
Nano-Chip is an easy « Plug and Play » system for highly
sensitive protein analysis
Polaris CHIP provides more qualitative information and
this should be transposed to quantitative analysis (QQQ)
Nano-CHIP should be used carefully (Qty of sample)
… To be continued
Agilent MS User Meeting 1st July 2014
Acknowledgements
Prof. J. Nève
Prof. M. Gelbcke
Prof. Ass. F. Dufrasne
Prof. Ass. P. Van Antwerpen
Prof. Ass. C. De Vriese
Analytical Platform of the Faculty of Pharmacy (ULB):
Prof. Ass. P. Van Antwerpen
M. Cortese
D. Dufour
Laboratory of Pharmaceutical Chemistry (ULB):
Dr G. Berger
Dr J. Soubhye
C. Noyon
A. Planinc
F. Reyé
I. Aldib
This study was supported by grants from the Belgian Fund for Scientific Research (F.R.S.-FNRS) and the FER – ULB.
Agilent Technologies
M. Haex
F. Abts
Agilent MS User Meeting 1st July 2014 28
Thank for your attention …
… and go on Red Devils
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