Isoelectric Focusing: Sample Pretreatment – Separation – Hyphenation
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Isoelectric Focusing: Sample Pretreatment – Separation – Hyphenation Linda Silvertand ISBN: 978-90-8891-113-2 Printing: www.proefschriftmaken.nl Copyright: ©2009 by Linda Silvertand Cover design: Linda Silvertand - Lak en anthraciet op canvas Niets uit deze uitgave mag verveelvoudigd en/of openbaar gemaakt worden zonder voorafgaande schriftelijke toestemming van de auteur. All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means without written permission of the author and the publisher holding the copyrights of the published articles. Isoelectric Focusing: Sample Pretreatment – Separation – Hyphenation Isoelectrisch Focusseren: Monstervoorbewerking - Scheiding - Koppeling (met een samenvatting in het Nederlands) Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof. dr. J.C. Stoof, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op woensdag 23 september 2009 des middags te 2.30 uur door Linda Henriette Hermina Silvertand geboren op 21 juni 1979 te Heerlen Promotor: prof. dr. G.J. de Jong Co-promotor: dr. W.P. van Bennekom This research is part of the IOP Genomics project (STW 06209) “Proteomics on a chip for monitoring auto-immune diseases” and is supported by the Netherlands Research Council for Chemical Sciences (NWO/CW) with financial aid from the Netherlands Technology Foundation (STW). The printing of this thesis was financially supported by: UIPS (Utrecht Institute for Pharmaceutical Sciences) Table of Contents Chapter 1 Introduction ... ……………………………………………………………..……………………….7 1.1 Project goals ..................................................................................................... 9 1.2 Introduction to (bio)analytical techniques ..................................................... 13 1.3 Outline of the thesis ....................................................................................... 25 Chapter 2 Recent advances in capillary isoelectric focusing 2003-2007…............31 2.1 Introduction .................................................................................................... 33 2.2 Methodology .................................................................................................. 34 2.3 Detection ........................................................................................................ 42 2.4 Multidimensional systems .............................................................................. 49 2.5 Microfluidic devices ........................................................................................ 52 2.6 Applications .................................................................................................... 53 2.7 Conclusion ....................................................................................................... 60 Chapter 3 Improved repeatability and MALDI-TOF MS compatibility in cIEF………67 3.1 Introduction .................................................................................................... 69 3.2 Materials and methods ................................................................................... 71 3.3 Results and discussion .................................................................................... 73 3.4 Concluding remarks ........................................................................................ 83 Chapter 4 Characterization of cIEF-MALDI-TOF MS for protein analysis .... ………… 89 4.1 Introduction .................................................................................................... 91 4.2 Materials and methods ................................................................................... 93 4.3 Results and discussion .................................................................................... 95 4.4 Concluding remarks ...................................................................................... 105 Chapter 5 Development and evaluation of an IEF-iSPR system for biomarker research ........ ……………………………………………………………………………….…111 5.1 Introduction .................................................................................................. 113 5.2 Materials and methods ................................................................................. 116 5.3 Results and discussion .................................................................................. 118 5.4 Concluding remarks ...................................................................................... 125 Chapter 6 Selective protein removal and desalting using microchip CE……………129 6.1 Introduction .................................................................................................. 131 6.2 Materials and methods ................................................................................. 133 6.3 Results and discussion .................................................................................. 135 6.4 Conclusion ..................................................................................................... 140 Chapter 7 Conclusions and recommendations ... ………………………………………………..145 7.1 Conclusion ..................................................................................................... 147 7.2 Recommendations ........................................................................................ 151 Samenvatting ......................................................................................................... 157 Abbreviations ......................................................................................................... 165 Curriculum Vitae .................................................................................................... 171 Publications ............................................................................................................ 175 Acknowledgements ............................................................................................... 181 Chapter 1 - Introduction Chapter 1 Introduction Page | 7 Chapter 1 - Introduction Page | 8 Chapter 1 - Introduction 1.1 Project goals The IOP Genomics project ‘Proteomics on a chip for monitoring auto-immune diseases’ was initiated by three universities: University of Twente, University of Utrecht and the University of Nijmegen, with the goal of developing separation and identification strategies for biomarkers (e.g. proteins and peptides) in highly complex biological matrices. Several industrial partners were also involved in the project and contributed scientifically as well as financially. The project was funded by the Netherlands Technology Foundation, STW. ‘Proteomics on a chip’ The goal of the total project was: ‘… to develop an integrated “proteomics on a chip” device which comprises two successive on-chip separations and solid-phase affinity binding combined with Surface Plasmon Resonance (SPR*) imaging’ [1]. The intent was to use this device for finding new biomarkers and to develop new diagnostic applications for auto-immune diseases in general, with emphasis on rheumatoid arthritis. To achieve this goal, each of the participating universities contributed with their own area of expertise (Fig. 1.1): Nijmegen Utrecht (Pharm Anal) Twente Utrecht (BioMS) Sample 1st dimension 2nd dimension Address flow- Sample SPR-MS clean-up separation separation SPR Miniaturization Proteomics on a chip Figure 1.1. Schematic overview of contributions of the different academic partners: University of Nijmegen (green), University of Utrecht (red) and University of Nijmegen (blue) to achieve “Proteomics on a chip”. • The University of Nijmegen (prof. dr. G.J.M. Pruijn) focuses on the pathogenesis of rheumatoid arthritis and other auto-immune diseases. Studying disease mechanisms, supplying and testing the relevant recombinant antigens, auto-antibodies and patient sera with the iSPR technology is their part in the project. • The Pharmaceutical Analysis group (prof. dr. G.J. de Jong and dr. W.P. van Bennekom) of the University of Utrecht is specialised in sample pretreatment, *) On page 165 is a list of abbreviations Page | 9 Chapter 1 - Introduction sensing and separation techniques. This expertise is to be used for developing clean-up and separation procedures and the coupling to sensing techniques. • The Biomolecular Mass Spectrometry group (prof. dr. A.J. Heck), was responsible for the last step in the project: mass spectrometric confirmation and identification and the hyphenation of SPR to MS. • The Biochip group (University of Twente, dr. ir. R.B.M. Schasfoort) has to integrate the developments of the other groups on microchip format and is responsible for the address-flow interface to iSPR. Project goals: clean-up, separation and hyphenation For the sample clean-up and separation part a literature study was carried out on how to approach the problem of isolating possible protein biomarkers from a highly complex matrix. From this survey, the project goals for this thesis were formulated. In proteomic applications, patient samples, like blood, synovial fluid, organ tissue, etc. often need to be analysed e.g. in the search for signalling proteins or marker proteins. The biological matrices are highly complex and contain next to the compound of interest a large number of substances like salts, lipids, small molecules, cells, etc., which can interfere with the separation and analysis of the biomarkers. In order to remove these compounds from the sample (reducing the sample complexity), an appropriate