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

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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

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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