Dr Cédric DELPORTE Prof. Ass. Dr Pierre VAN ANTWERPEN

Transcription

1 POLARIS CHIP FOR PROTEOMIC S: PROTEIN AND PEPTIDE PROFILING Dr Cédric DELPORTE Prof. Ass. Dr Pierre VAN ANTWERPEN Laboratory of Pharmaceutical Chemistry & Analytical Platform of the Faculty of Pharmacy (ULB)

2 Overview 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 2

3 Introduction Atherosclerosis Inflammatory disease Oxidized LDLs (oxldls) Myeloperoxidase (MPO) MoxLDLs HOCl H 2 O 2, Cl - 3/16

4 Introduction Atherosclerosis Primary granule LDL Oxidation PMN Neutrophil MPO Nat-LDL H2O2 HOX SOD AT II 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 Mox-LDL TNFα O2 - Endothelial cell IL-8 NOX2 AT-R1 H2O2 Nat-LDL Mox-LDL Monocyte/ Macrophage Intima Foam Cell Scavenger receptor Agilent User Meeting 1st July Delporte et al,ms Mediators of inflammation 4

5 Example of Apolipoprotein B100 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 5

6 Example of Apolipoprotein B100 Steps: 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 Spectum Mill 6

7 Example of Apolipoprotein B % of sequence recovery Validation of false-positive peptides Default automatic validation + manual validation 7

8 Overview 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 8

9 Example of Apolipoprotein B100 4 fold higher Modified peptides (PTMs) 9

10 Example of Apolipoprotein B100 LDL oxidation in vitro by MPO vs HOCl Detection of MPO-specific oxidation: MPOsyst 50 and 100 µm MPOsyst 100 µm Chemical Oxidation Enzymatic Oxidation * To mimic MPO/H 2 O 2 /Cl - Oxidation MPO (MoxLDL) 10

11 Example of Apolipoprotein B100 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

12 Example of Apolipoprotein B100, Polaris CHIP Nano LC-Chip Cube MS system 12

13 Example of Apolipoprotein B100, Polaris CHIP Stator Autosampler Waste Nanopump Side View Rotor inner rotor Rotor outer rotor Stator Microvalve HPLC-Chip 13

14 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

15 Example of Apolipoprotein B100, Polaris CHIP CHIP characteristics Prot Id-CHIP 150 Polaris HR CHIP Phase, chemistry Zorbax C18 Polaris C18 Particule size 5 µm 3 µm Porosity 300 Å Increase resolution 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 µl/min (0.4) µl/min (0.3)

16 Example of Apolipoprotein B100, Polaris CHIP 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

17 Overview 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 17

18 Atheromatous plaque Plaques (aorta, carotids or abdominal artery) are extracted from patients The tissues are frozen and crushed 18

19 Experimental Only delipidation (A) vs Extraction + Delipidation (B) A 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 2.5 g of powder B 4 ml of Extraction buffer Extraction 24h at 4 C (Rotamix) (Twice) Deplipidation of 1 ml extract Reduction, Alkylation and Digestion by trypsin (24h) Extraction Buffer = 0.15 M NaCl, 100 µm DTPA 100 µm BHT, protease inhibitor mixture (50 µl/4 ml), 10 mm phosphate, ph /16

20 Experimental LC conditions Injection Volume: 5 µl RRLC Poroshell 120 EC-C x 100 mm, 2.7 µm Gradient Time [min] % B Flow [ml/min] Stop Time /16

21 Experimental LC conditions Injection Volume: 5 µl ProtID-Chip-150 (II) 300 Å C x 150 mm, 40 nl trap column Large capacity Chip (II) High Capacity Loading, 300 Å C x 150 mm, 160 nl trap column Polaris-HR-Chip 3C18High Resolution Chip 150 mm 180 Å 3 µm C18, x 150 mm, 360 nl trap column Gradient Time [min] % B Gradient Time [min] % B Gradient Time [min] % B 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 21/16

22 Atheromatous plaque Number of spectra Total witout abundant proteins RRLC Large Cap ProteinID 150 Polaris Lots of abundant proteins are found such as albumin, fibrinogen, immunoglubulins and hemoglobin Total 197 witout abundant proteins Number of unique peptides Total witout abundant proteins Total Found Proteins RRLC Large Cap ProteinID 150 Polaris RRLC Large Cap ProteinID 150 Polaris

23 Atheromatous plaque Total witout abundant proteins Total Found Proteins RRLC Large Cap ProteinID 150 Polaris Lots of abundant proteins are found such as albumin, fibrinogen, immunoglubulins and hemoglobin Protein Name Serum albumin Serotransferrin Fibrinogen beta chain Hemoglobin subunit beta Hemoglobin subunit delta Hemoglobin subunit gamma-1 Alpha-1-antitrypsin Alpha-2-macroglobulin Actin, alpha cardiac muscle 1 Actin, cytoplasmic 2 Beta-actin-like protein 2 Ig gamma-2 chain C region Ig gamma-1 chain C region Ig gamma-3 chain C region Apolipoprotein A-I Haptoglobin Ig mu chain C region Complement C3 Fibrinogen gamma chain Ig alpha-1 chain C region Fibrinogen alpha chain Hemoglobin subunit alpha Protein_ID P02768 P02787 P02675 P68871 P02042 P69891 P01009 P01023 P68032 P63261 Q562R1 P01859 P01857 P01860 P02647 P00738 P01871 P01024 P02679 P01876 P02671 P

24 Atheromatous plaque Protein Id Pept number RRLC Pept numb CHIP Apolipoprotein A-I Apolipoprotein A-II 0 2 Apolipoprotein A-IV 2 2 Apolipoprotein B Apolipoprotein E 4 5 Total 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 24

25 Overview 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 25

26 Perspectives and conclusions 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 26

27 Acknowledgements Laboratory of Pharmaceutical Chemistry (ULB): Prof. J. Nève Prof. M. Gelbcke Prof. Ass. F. Dufrasne Prof. Ass. P. Van Antwerpen Prof. Ass. C. De Vriese Dr G. Berger Dr J. Soubhye C. Noyon A. Planinc F. Reyé I. Aldib Analytical Platform of the Faculty of Pharmacy (ULB): Prof. Ass. P. Van Antwerpen M. Cortese D. Dufour Agilent Technologies M. Haex F. Abts This study was supported by grants from the Belgian Fund for Scientific Research (F.R.S.-FNRS) and the FER ULB.

28 Thank for your attention and go on Red Devils 28