Développement de méthodes chromatographiques liquides

De
Publié par

Niveau: Supérieur

  • dissertation


Développement de méthodes chromatographiques liquides multidimensionnelles couplées à la spectrométrie de masse, préparation et analyse d'échantillons biologiques complexes ------------------------ Development of multidimensional liquid chromatographic methods hyphenated to mass spectrometry, preparation and analysis of complex biological samples Thèse présentée pour l'obtention du grade de DOCTEUR DE L'UNIVERSITE LOUIS PASTEUR DE STRASBOURG par Nathanaël DELMOTTE Soutenue le 12 juillet 2007 devant la commission d'examen : Dr. Alain VAN DORSSELAER Directeur de thèse Prof. Christian HUBER Co-directeur de thèse Prof. Marie-Claire HENNION Rapporteur externe Prof. Rolf MÜLLER Rapporteur externe Prof. Laurence SABATIER Rapporteur interne Dr. Andreas THOLEY Examinateur

  • spectrométrie de masse

  • dorsselaer durchgeführt

  • van dorsselaer

  • doktors der

  • laboratoire de spectrométrie de masse bio-organique

  • alain van

  • instrumentelle analytik und

  • dr. alain

  • der universität des saarlandes


Publié le : dimanche 1 juillet 2007
Lecture(s) : 101
Source : scd-theses.u-strasbg.fr
Nombre de pages : 205
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Développement de méthodes chromatographiques liquides
multidimensionnelles couplées à la spectrométrie de masse,
préparation et analyse d’échantillons biologiques complexes

------------------------

Development of multidimensional liquid chromatographic
methods hyphenated to mass spectrometry, preparation
and analysis of complex biological samples





Thèse


présentée pour l’obtention du grade de


DOCTEUR DE L’UNIVERSITE LOUIS PASTEUR

DE STRASBOURG


par

Nathanaël DELMOTTE



Soutenue le 12 juillet 2007 devant la commission d’examen :

Dr. Alain VAN DORSSELAER Directeur de thèse
Prof. Christian HUBER Co-directeur de thèse
Prof. Marie-Claire HENNION Rapporteur externe
Prof. Rolf MÜLLER Rapporteur externe
Prof. Laurence SABATIER Rapporteur interne
Dr. Andreas THOLEY Examinateur



Development of multidimensional liquid chromatographic
methods hyphenated to mass spectrometry, preparation
and analysis of complex biological samples






Dissertation





zur Erlangung des Grades
des Doktors der Naturwissenschaften
der Naturwissenschaftlich-Technischen Fakultät III
Chemie, Pharmazie und Werkstoffwissenschaften
der Universität des Saarlandes





von

Nathanaël DELMOTTE








Saarbrücken

2007




















Tag des Kolloquiums : 12. Juli 2007

Dekan : Prof. Dr. Uli Müller

Mitglieder des
Prüfungsausschusses : Prof. Dr. Christian Huber
Dr. Alain Van Dorsselaer
Prof. Dr. Rolf Müller
Prof. Dr. Laurence Sabatier Marie-Claire Hennion
Dr. Andreas Tholey



This doctoral thesis was performed under the joint supervision of Prof.
Dr. Christian Huber and Dr. Alain Van Dorsselaer.

Ce travail de thèse a été réalisé dans le cadre d’une co-tutelle sous la
direction du Prof. Dr. Christian Huber et du Dr. Alain Van Dorsselaer.

Die vorliegende Dissertation wurde unter der Leitung von Prof. Dr.
Christian Huber und Dr. Alain Van Dorsselaer durchgeführt.













Prof. Dr. Christian HUBER
Instrumentelle Analytik und Bioanalytik
Universität des Saarlandes
Postfach 15 11 50
D-66041 Saarbruecken
Tel: +49 (0) 681 302 3433 Fax: 49 (0) 681 302 2963 E-mail: christian.huber@mx.uni-saarland.de


Dr. Alain VAN DORSSELAER
Institut Pluridisciplinaire Hubert Curien
Sciences Analytiques et Interactions Ioniques et Biomoléculaires
Laboratoire de Spectrométrie de Masse Bio-Organique
Université Louis Pasteur
ECPM, 25 Rue Becquerel, F- 67087 Strasbourg Cedex 2
Tel: +33 (0) 3 90 24 27 83 Fax: +33 (0) 3 90 24 27 81 E-mail: vandors@chimie.u-strasbg.fr


5




















































Acknowledgements
First of all, I would like to thank my both supervisors, Prof. Dr. Christian Huber and
Dr. Alain Van Dorsselaer for giving me the opportunity to graduate in their lab. I am grateful
for the guidance and support. Herzlichen Dank!

Prof. Dr. Laurence Sabatier, Prof Dr. Marie-Claire Hennion, Prof Dr. Rolf Müller, and
PD Dr. Andreas Tholey are acknowledged for accepting to spend some of their precious
time to correct and evaluate this work.

I also want to thank Prof. Dr. Dr. h.c. Heinz Engelhardt, PD Dr. Frank Steiner, and
Dr. Markus Martin for their extremely valuable answers to any of my questions.

I am grateful to Dr. Uwe Kobold, Dr. Thomas Meier, Dr. Andreas Gallusser, Dr. Albert
Geiger, Thomas Dülffer, Sabine Kerkenbusch, and Thomas Weidner from Roche
Diagnostics for the financial support as well as for their assistance, input, and advice.

My sincere thanks go to Reiner Wintringer, “Windy”. Without him, the instruments would not
be running so good, and the everyday life in the lab would not be so peaceful!

I kindly thank Christa Göllen, Gabriele Krug, and Véronique Trimbour for their help in the
intricacies of the German and French red tape!

I warmly thank my first colleagues Dr. Bettina Mayr, Dr. Hansjörg Toll, and Dr. Christian
Schley for their good advice, shrewdness, and tricks.

My very special thanks go to Maria Lasaosa for the very pleasant (and successful!) team-
working, and Prof. Dr. Elmar Heinzle for permitting this cooperation.

I also want to thank my “Hiwi-Team”: Silke Ruzek, Volker Neu, and Thomas Jakoby, as
well as Benny Kneissl for the useful bioinformatics software.

Of course (!) I also thank Verena Fraaß, Andreas Leinenbach, Katja Melchior, and Jens
Mohr. I cannot forget Dr. Anis Mahsunah, Eva Luxenburger, Iris Gostomski, Manuela
Hügel, Rainer Geiss, Devid Hero, Bilgin Vatansever, Dr. Leena Suntornsuk, Sascha
Quinten, Patrick Riefer, and Nathalie Selevsek. I am grateful for all their encouragements,
futile discussions, and jokes; I really enjoyed the friendly work atmosphere!

Many thanks go to Dr. Christine Schaeffer and Dr. Jean-Marc Strub for their help and
assistance during my stays in Strasburg.

Thanks to Hans-Peter Skohoutil, Norbert Ochs, Jens Wiegert, and Robin Adolph for the
manufacturing and supply of many un-purchasable hardware!

My thanks go to Dionex, LCPackings and Bruker Daltonics for the instruments and to
Dr. Aleš Štrancar from BIA Separations for the CIM disks.

Last but not least, my hearty thanks go to the surrounding of my family and friends for their
joyful and constant support!
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Table of contents

Acknowledgements................................................................................7
Table of contents....................................................................................9
Table of abbreviations, acronyms, and symbols ..............................15
Preliminaries.........................................................................................17
English version........................................................................................................ 18
Version française .................................................................................................... 19
Résumé substantiel ................................................................................................ 20
Deutsche Version.................................................................................................... 22

Chapter I: Goal of the thesis ...............................................................23

Chapter II: Theoretical part..................................................................27
1 Biological samples............................................28
1.1 Biological fluids......................................................................................... 28
1.2 Cells and protein cell extracts .................................................................. 30
2 Structure and properties of proteins............................................32
2.1 Amino acids, peptides and proteins ......................................................... 32
2.2 Roles of proteins....................................................................................... 36
2.3 Proteome .................................................................................................. 37
3 High-performance liquid chromatography for the separation of
biomolecules ........................................................................................40
3.1 Reversed-phase and ion-pair reversed-phase HPLC.............................. 42
3.2 Ion-exchange HPLC ................................................................................. 44
3.3 Affinity chromatography............................................................................ 46
3.4 Size-exclusion chromatography ............................................................... 47
3.5 Restricted access materials ..................................................................... 48
4 Methods of detection .....................................................................50
4.1 Immunodetection methods....................................................................... 50
4.2 Mass spectrometry ................................................................................... 51
9
4.2.1 Principle of electrospray ionization........................................................... 51
4.2.2 Quadrupole mass analyzer ...................................................................... 54
4.2.3 ole ion trap mass analyzer ......................................................... 55
4.3 Identification of peptides and proteins with mass spectrometry and
algorithmic computation .......................................................................................... 58

Chapter III: Development of monolithic immuno-adsorbers for the
isolation of biomarkers from human serum ......................................61
1 Introduction ....................................................................................62
1.1 Aim of the work......................................................................................... 62
1.2 Investigated biomarkers ........................................................................... 64
1.2.1 Myoglobin as biomarker of heart infarct................................................... 64
1.2.2 NT-proBNP as biomarker of heart insufficiency....................................... 65
2 Materials and methods ..................................................................66
2.1 Chemicals and samples ........................................................................... 66
2.2 Analytical setups....................................................................................... 67
2.2.1 Isolation of biomarkers with CIM disks..................................................... 67
2.2.2 High-performance liquid chromatography-mass spectrometry................ 67
2.2.3 Detection and quantitation of NT-proBNP with immunoassays............... 69
2.3 Preparation of affinity CIM disks .............................................................. 70
2.3.1 tion of anti-myoglobin- and anti-NT-proBNP CIM disks via direct
immobilization ......................................................................................................... 70
2.3.2 Preparation of affinity CIM disks via streptavidin-biotin anchorage ......... 71
3 Isolation of myoglobin from human serum by affinity
chromatography...................................................................................74
3.1 Isolation of myoglobin at high concentration............................................ 74
3.2 of myoglobin at low concentration ............................................. 76
3.3 Conclusions .............................................................................................. 78
4 Isolation of NT-proBNP from human serum by affinity
chromatography...................................................................................79
4.1 Evaluation of the loadability of anti-NT-proBNP-CIM disk ....................... 79
4.2 Isolation of NT-proBNP from human serum at 125 fmol/µL..................... 80
4.3 of NT-proBNP serum at 7.8 fmol/µL...................... 82

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