MASS SPECTROMETRY AND GENOMIC ANALYSIS

Transcription

1 MASS SPECTROMETRY AND GENOMIC ANALYSIS

2 FOCUS ON STRUCTURAL BIOLOGY Volume 2 Series Editor ROB KAPTEIN Bijvoet Center for Biomolecular Research, Utrecht University, The Netherlands

3 Mass Spectrometry and Genomic Analysis Edited by J. NICHOLAS HOUSBY Oxagen Limited, Abingdon, United Kingdom KLUWER ACADEMIC PUBLISHERS NEW YORK, BOSTON, DORDRECHT, LONDON, MOSCOW

4 ebook ISBN: Print ISBN: Kluwer Academic Publishers New York, Boston, Dordrecht, London, Moscow Print 2001 Kluwer Academic Publishers Dordrecht All rights reserved No part of this ebook may be reproduced or transmitted in any form or by any means, electronic, mechanical, recording, or otherwise, without written consent from the Publisher Created in the United States of America Visit Kluwer Online at: and Kluwer's ebookstore at:

5 DEDICATION I would like to dedicate this book to my wife Carol, for her love, support and encouragement, as always.

6 TABLE OF CONTENTS INTRODUCTION PREFACE TJ. Griffin, LM. Smith CHAPTER 1 xiii xv Single-Nucleotide Polymorphism Analysis by MALDI-TOF Mass Spectrometry 1. Introduction 1.1. MALDI-TOF MS Analysis of Peptide Nucleic Acid Hybridisation Probes 2.1. Design of PNA Hybridisation Probes Analysis of Polymorphisms in Tyrosinase Exon Direct Analysis of Invasive Cleavage Products The Invader Assay Direct Analysis of SNPs From Human Genomic DNA 8 Conclusions Experimental Methods 5.1. PNA Probe Synthesis and Preparation 5.2. PCR Amplification of Exon 4 of the Tyrosinase Gene 5.3. Hybridisation of PNA Probes to Immobilised Gene Targets 5.4. MALDI-TOF MS Analysis of PNA Probes 5.5. Invader Squared Reaction 5.6. MALDI-TOF MS Sample Preparation of Cleavage Products 5.7. MALDI-TOF MS Analysis of Cleavage Products 6. Affiliations 7. References CHAPTER 2 LA. Haff, AC. Belden, LR. Hall, PL. Ross, IP. Smirnov SNP Genotyping by MALDI-TOF Mass Spectrometry 1. Introduction 2. SNP Analysis by Single Base Extension of Primers 3. Materials and Methods 4. Design Considerations for the SNP Genotyping Assay 4.1. Design of PCR Product 4.2. PCR Product Polishing 4.3. Primer Design Rules for Monoplex SNP Typing vii

7 viii 4.4. Mass Calculations Primer Design Rules for Multiplexed Reactions Multiplexing with Primer Pools of Six or Fewer Primers Recommended Primer Pool Design: More Than Six Primers Primer Quality The Single Base Extension Reaction Desalting of Primer Extension Reactions MALDI-TOF Conditions Determination of Bases Added to the Primer Modification of the SNP Typing Assay to Support Allele Frequency Determination Conclusions References 32 Hubert Köster CHAPTER 3 MASSARRAY : Highly Accurate and Versatile High Throughput Analysis of Genetic Variations Introduction MassARRAY Technology Methodology of MassARRAY Technology Diagnostic Applications of MassARRAY Technology for Analysis of DNA Sequence Variations Application of MassARRAY for Confirmation and Validation of Single Nucleotide Polymorphisms Conclusions 7. Materials and Methods 8. Acknowledgements 9. References S. Sauer, D. Lechner, IG. Gut The GOOD Assay CHAPTER 4 1. Introduction SNP Genotyping by MALDI How to Improve the Analysis of DNA by MALDI Principles of the GOOD Assay 54

8 ix 5. Variations of the GOOD Assay Materials and Method of the GOOD Assay Applications of the GOOD Assay The Issue of DNA Quality Physical Haplotyping by the GOOD Assay Quantitation Automation of the GOOD Assay Outlook References 65 CHAPTER 5 PH. Tsatsos, V. Vasiliskov, A. Mirzabekov Microchip Analysis of DNA Sequence by Contiguous Stacking of Oligonucleotides and Mass Spectrometry 1. Introduction Magichip properties Production of MAGIChip Activation of Probes Chemical Immobilisation of Probes Preparation of the Target Hybridisation Theoretical Considerations of Hybridisation Hybridisation on Microchips Generic Microchip Principle of Contiguous Stacking Hybridisation Monitoring Fluorescence Laser Scanner Mass Spectrometry Example of Mutation Detection by CSH and MALDI-TOF Mass Spectrometry Conclusions Acknowledgements References 74 CHAPTER 6 PE. Jackson, MD. Friesen, JD. Groopman Short Oligonucleotide Mass Analysis (SOMA): an ESI-MS Application for Genotyping and Mutation Analysis

9 x 1. Introduction Short Oligonucleotide Mass Analysis Method Outline Design of PCR Primers and Fragments for Analysis Typical PCR Reaction Conditions Electrospray Ionisation Mass Spectrometry Formation of Ions Tandem Mass Spectrometry Typical ESI-MS Settings for SOMA Purification Procedures Phenol/Chloroform Extraction and Ethanol Precipitation In-line HPLC Purification Genotyping Using SOMA APC Genotyping in Human Subjects APC Genotyping in Min Mice Mutation Detection Using SOMA Analysis of p53 Mutations in Liver Cancer Patients p53 Mutations in Liver Tumours p53 Mutations in Plasma Samples Advantages and Disadvantages of SOMA Future Perspectives Acknowledgements References 91 CHAPTER 7 WV. Bienvenut, M. Müller, PM. Palagi, E. Gasteiger, M. Heller, E. Jung, M. Giron, R. Gras, S. Gay, PA. Binz, G J. Hughes, JC. Sanchez, RD. Appel, DF. Hochstrasser Proteomics and Mass Spectrometry: Some Aspects and Recent Developments 1. Introduction to Proteomics Protein Biochemical and Chemical Processing Followed by Mass Spectrometric Analysis DE Gel Protein Separation 2.2. Protein Identification Using Peptide Mass Fingerprinting and Robots MALDI-MS Analysis MS/MS Analysis Improvement of the Identification by Chemical Modification of Peptides 2.3. The Molecular Scanner Approach Double Parallel Digestion Process Quantitation of the Transferred Product and Diffusion Protein Identification Using Bioinformatics Tools 3.1. Protein Identification by PMF Tools Using MS Data

10 xi Peak Detection Identification Tools MS/MS Ions Search De Novo Sequencing Other Tools Related to Protein Identification Data Storage and Treatment with LIMS Concluding Remarks Bioinformatics Tools for the Molecular Scanner Peak Detection and Spectrum Intensity Images Protein Identification Validation of Identifications Concluding Remarks Conclusions Acknowledgements References 141 INDEX 147

11 INTRODUCTION The human genome project has created intense interest from academics, commercial business and, not least, the general public. This is not surprising, as understanding the genetic make up of each individual gives us clues as to the genetic factors that predispose one to a particular genetic disease. In this way the human genome sequence is set to revolutionise the way we treat people for genetic diseases and/or predict patients future health regimes. Single Nucleotide Polymorphisms (SNPs), single base changes in the nucleotide DNA sequence of individuals, are thought to be the main cause of genetic variation. It is this variation that is so exciting as it underpins the way(s) in which the human body can respond to drug treatments, natural defence against disease susceptibility or the stratification of the disease in terms of age of onset or severity. These SNPs can be either coding (csnp), appearing within coding regions of genes or in areas of the genome that do not encode for proteins. The coding csnps may alter the amino acid protein sequence which in turn may alter the function of that particular protein. Much effort is directed towards identifying the functions of SNPs, whether that be within genes (csnps) or within regulatory regions (eg. promoter region) that affect the level of transcription of the gene into mrna. If an SNP is proved to be truly polymorphic, i.e. it appears in many samples of the population, then individuals can be genotyped for the homozygous form of the allele, the same variation on both chromosomes, or a heterozygous form with a different variation of the SNP on each chromosome. An international SNP working group has been set up to map all of the known human SNPs, it is envisaged that every single gene in the human genome will have a variation within or close to it. By comparing patterns of SNP allele frequencies between disease affected and control populations, disease associated SNPs can be identified and potential disease gene(s) located. These types of study require genotyping of thousands of SNPs which requires the use of powerful, high throughput, systems of analysis. There are many competing new technology platforms which attempt this but the one that stands out from the crowd is mass spectrometry. This book contains a collection of descriptions of some of the most outstanding advances in this field of mass spectrometry (chapters 1-6), from which, I hope, the reader will be able to learn both the principles and the most up to date methods for its use. Analysis of the proteins produced from mrna will lead to another level of information analysis. Not all of the proteins produced from mrna correlates to its expression. Many proteins have alterations at the post-translational stage, mostly by glycosylation or phosphorylation events. It is this that may cause alteration in function of the protein product. It is therefore necessary to investigate at both the gene level and at the protein level. The study of proteomics, the comprehensive study of proteins in a given cell, is discussed in chapter 7. This gives the reader a broader perspective in the uses of mass spectrometry in this fast changing analytical environment of genome research. xiii J. NICHOLAS HOUSBY

12 PREFACE My interest in mass spectrometry stemmed from working in the laboratory of Professor Edwin Southern at the department of Biochemistry, Oxford University, UK. It was there that I was given an ambitious project which involved the analysis of arrays of nucleic acids using mass spectrometry. I must certainly thank him for his tremendous insights into this field and for stimulating my interest in this area of research. Having now moved on from Professor Southern s lab I have become extremely interested in the use of novel technologies for genetic analysis. I am convinced, that over the next decade, mass spectrometry will lead the way in polymorphism screening, genotyping and in other genetic testing environments. It is for this reason that I have put together this book. I have attempted to bring together descriptions, from some of the world leaders in this field of research, of the most recent advances in genomic analysis using mass spectrometry. I make no attempt to make this an exhaustive collection but a text that will whet the appetite of those interested in this fast moving and provocative arena. The final chapter describes the use of mass spectrometry in proteomics, the comprehensive (high throughput) study of proteins in cells. I think that this is a necessary addition for the reader to have a broader insight into the current uses of mass spectrometry in research and development. I hope that this book will be a useful companion to investigators already at the cutting edge but also a guide to those who are interested in learning more about this powerful analytical tool. J. NICHOLAS HOUSBY xv