IDENTIFICATION OFMICROORGANISMS BYMASS SPECTROMETRY

Edited by

CHARLES L. WILKINSJACKSON O. LAY, JR.University of Arkansas, Fayetteville, AR

A JOHN WILEY & SONS, INC., PUBLICATION

Innodata0471748633.jpg

IDENTIFICATION OF MICROORGANISMS BY MASS SPECTROMETRY

CHEMICAL ANALYSIS

A SERIES OF MONOGRAPHS ON ANALYTICAL CHEMISTRY AND ITS APPLICATIONS

Edited byJ. D. WINEFORDNER

VOLUME 169

IDENTIFICATION OFMICROORGANISMS BYMASS SPECTROMETRY

Edited by

CHARLES L. WILKINSJACKSON O. LAY, JR.University of Arkansas, Fayetteville, AR

A JOHN WILEY & SONS, INC., PUBLICATION

Copyright 2006 by John Wiley & Sons, Inc. All rights reserved.

Published by John Wiley & Sons, Inc., Hoboken, New Jersey.Published simultaneously in Canada.

No part of this publication may be reproduced, stored in a retrieval system, or transmitted inany form or by any means, electronic, mechanical, photocopying, recording, scanning, orotherwise, except as permitted under Section 107 or 108 of the 1976 United States CopyrightAct, without either the prior written permission of the Publisher, or authorization throughpayment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222Rosewood Drive, Danvers, MA 01923, 978-750-8400, fax 978-646-8600, or on the web at

Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030,

efforts in preparing this book, they make no representations or warranties with respect to theaccuracy or completeness of the contents of this book and specifically disclaim any impliedwarranties of merchantability or fitness for a particular purpose. No warranty may be createdor extended by sales representatives or written sales materials. The advice and strategiescontained herein may not be suitable for your situation. You should consult with a professionalwhere appropriate. Neither the publisher nor author shall be liable for any loss of profit or anyother commercial damages, including but not limited to special, incidental, consequential, orother damages.

For general information on our other products and services please contact our Customer Care Department within the U.S. at 877-762-2974, outside the U.S. at 317-572-3993 or fax 317-572-4002.

Wiley also publishes its books in a variety of electronic formats. Some content that appears inprint may not be available in electronic format. For more information about Wiley products,

Library of Congress Cataloging-in-Publication Data:

Identification of microorganisms by mass spectrometry / edited by Charles L. Wilkins,Jackson O. Lay, Jr.

p. cm.Includes bibliographical references and index.ISBN-13 978-0-471-65442-1 (cloth)ISBN-10 0-471-65442-6 (cloth)

1. MicroorganismsIdentification. 2. Mass spectrometry. I. Wilkins, Charles L.(Charles Lee), 1938 II. Lay, Jackson O.

QR67.I344 2006579dc22 2005008550

Printed in the United States of America

10 9 8 7 6 5 4 3 2 1

(201) 748-6011, fax (201) 748-6008 or online at http://www.wiley.com/go/permission.

visit our web site at www.wiley.com.

Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best

www.copyright.com. Requests to the Publisher for permission should be addressed to the

http://www.copyright.comhttp://www.wiley.com/go/permissionhttp://www.wiley.com

CONTENTS

v

Preface xi

Contributors xv

1 Cultural, Serological, and Genetic Methods for Identification of Bacteria 1John B. Sutherland and Fatemeh Rafii

1.1 Introduction, 11.2 Identification of Bacteria by Cultural Methods, 31.3 Identification of Bacteria by Serological Methods, 61.4 Identification of Bacteria by Genetic Methods, 81.5 Other Methods Used for Bacterial Characterization, 121.6 Conclusions, 13Acknowledgments, 13References, 13

2 Mass Spectrometry: Identification and Biodetection, Lessons Learned and Future Developments 23Alvin Fox

2.1 Introduction, 232.2 Analysis of Fatty Acid and Sugar Monomers by GC-FID,

GC-MS, and GC-MS-MS, 242.3 Analysis of PCR Products by PCR, PCR-MS, and

PCR-MS-MS, 26

2.4 Analysis of Proteins by MALDI-TOF MS, 312.5 Chemical Markers for Protein-Based Identification or

Biodetection, 322.6 Conclusions, 33References, 34

3 An Introduction to MALDI-TOF MS 39Rohana Liyanage and Jackson O. Lay, Jr.

3.1 Introduction, 393.2 Mass Spectrometry and Time-of-Flight MS, 403.3 Matrix-Assisted Laser Desorption Ionization, 473.4 MALDI-TOF Mass Spectrometry, 513.5 MALDI-TOF and Bacterial Identification, 513.6 Conclusions, 55References, 56

4 The Development of the Block II Chemical Biological Mass Spectrometer 61Wayne H. Griest and Stephen A. Lammert

4.1 Introduction, 614.2 Development History and Design Philosophy, 634.3 Requirements and Specifications, 734.4 Performance Testing, 794.5 Conclusions, 86Acknowledgments, 87References, 87

5 Method Reproducibility and Spectral Library Assembly for Rapid Bacterial Characterization by Metastable Atom Bombardment Pyrolysis Mass Spectrometry 91Jon G. Wilkes, Gary Miertschin, Todd Eschler, Les Hosey,Fatemeh Rafii, Larry Rushing, Dan A. Buzatu, and Michel J. Bertrand

5.1 Introduction, 915.2 Sample Preparation for Rapid, Reproducible Cell Culture, 955.3 Analytical Instrumentation for Sensitive Detection and

Spectral Reproducibility, 1035.4 Spectral Library Assembly, 1085.5 Pattern Recognition Methods for Objectively Classifying

Bacteria, 1115.6 Conclusions, 120Acknowledgment, 121References, 121

vi CONTENTS

6 MALDI-TOF Mass Spectrometry of Intact Bacteria 125Jackson O. Lay, Jr., and Rohana Liyanage

6.1 Introduction, 1256.2 MALDI MS of Cellular Extracts, 1276.3 Taxonomy: From Isolates to Whole-Cell MALDI, 1296.4 Whole-Cell MALDI MS, 1316.5 Biology-Based Changes in Whole-Cell MALDI Spectra, 1336.6 Analysis of Mixtures, 1346.7 Experimental Approaches, 1366.8 Identification of Protein Markers, 1416.9 Detection of Target Proteins, 1426.10 Analysis of Clinical Isolates, 1436.11 Conclusions, 147References, 147

7 Development of Spectral Pattern-Matching Approaches to Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry for Bacterial Identification 153Kristin H. Jarman and Karen L. Wahl

7.1 Introduction, 1537.2 MALDI MS Signature Library Construction and

Identification, 1547.3 Full Spectrum Pattern Methods, 1557.4 Peak Table Based Methods, 1567.5 Future Directions, 158References, 159

8 Studies of Malaria by Mass Spectrometry 161Plamen A. Demirev

8.1 Introduction, 1618.2 Plasmodium in Red Blood Cells, 1638.3 Experimental Protocols for LDMS Detection of Malaria, 1648.4 Malaria Detection by Laser Desorption Mass Spectrometry, 1678.5 MS-Based Proteomics of the Plasmodium Parasite, 1748.6 Conclusions, 175Acknowledgments, 176References, 176

9 Bacterial Strain Differentiation by Mass Spectrometry 181Randy J. Arnold, Jonathan A. Karty, and James P. Reilly

9.1 Introduction, 1819.2 Analysis of Cellular Proteins by Mass Spectrometry, 182

CONTENTS vii

9.3 Application of MALDI-TOF to Bacteria Identification, 1839.4 Conclusions, 197Acknowledgment, 197References, 197

10 Bacterial Protein Biomarker Discovery: A Focused Approach to Developing Molecular-Based Identification Systems 203Tracie L. Williams, Steven R. Monday, and Steven M. Musser

10.1 Introduction, 20310.2 Protein Extraction Methods, 20610.3 Protein Chromatography, 20610.4 Mass Spectrometry, 21010.5 Automating the Process, 21210.6 Collecting and Sequencing Proteins, 21610.7 Conclusions, 222References, 222

11 High-Throughput Microbial Characterizations Using Electrospray Ionization Mass Spectrometry and Its Role in Functional Genomics 229Seetharaman Vaidyanathan and Royston Goodacre

11.1 Introduction, 22911.2 Microbial Characterizations beyond the Genomic Level:

Functional Genomics, 23011.3 Electrospray (Ionization) Mass Spectrometry (ESMS), 23311.4 ESMS of Microbes, 23711.5 Direct Infusion ESMS of Crude Cell extracts for

High-Throughput CharacterizationsMetabolic Fingerprinting and Footprinting, 247

11.6 Conclusions, 249Acknowledgment, 250References, 250

12 Bioinformatics for Flexibility, Reliability, and Mixture Analysis of Intact Microorganisms 257Catherine Fenselau and Patrick Pribil

12.1 Introduction, 25712.2 Library Matching, 25912.3 Machine Learning, 25912.4 Bioinformatics, 25912.5 Protein Molecular Masses, 26212.6 Peptide Maps, 265

viii CONTENTS

12.7 Microsequences from Peptides and Proteins, 26512.8 Remaining Challenges, 26912.9 Conclusions, 270Acknowledgments, 270References, 270

13 MALDI-FTMS of Whole-Cell Bacteria 279Jeffrey J. Jones, Michael J. Stump, and Charles L. Wilkins

13.1 Introduction, 27913.2 Fundamentals of MALDI-FTMS, 28013.3 Fundmentals of Complex Biological Analysis, 28213.4 Whole-Cell Characterization through MALDI-FTMS, 28313.5 Recombinant Overexpressed Proteins Desorbed Directly

from Whole Cells, 29313.6 Conclusions, 295References, 296

14 A Review of Antibody Capture and Bacteriophage Amplification in Connection with the Direct Analysis of Whole-Cell Bacteria by MALDI-TOF-MS 301Kent J. Voorhees and Jon C. Rees

14.1 Introduction, 30114.2 Bacterial Identification, 30314.3 Immunocapture of Bacterial Mixtures, 30814.4 Bacteriophage Amplification of Bacteria, 31114.5 Conclusions, 315References, 316

15 Discrimination and Identification of Microorganisms by Pyrolysis Mass Spectrometry: From Burning Ambitions to Cooling EmbersA Historical Perspective 319adaoin Timmins and Royston Goodacre

15.1 Introduction to Microbial Characterization, 31915.2 Principles of PyMS, 32315.3 Early Developments and Investigations, 1952 to 1985, 32515.4 The mid-1980s and Beyond, 32715.5 The Move from Cluster Analyses to Neural Networks, 33015.6 Reproducibility of PyMS, 332Acknowledgment, 334References, 334

Index 345

CONTENTS ix

PREFACE

The analysis of microorganisms by mass spectrometry is relatively new bymass spectrometry standards, having begun some 60 years after the firstreports of mass spectrometry studies in the last years of the nineteenth centuryby J. J. Thomson and others. Nevertheless, from a modern perspective, it could be argued that mass spectrometrists have been trying to sell microbiol-ogists (and others) on the idea of classifying bacteria using an instrumentalapproach since at least the 1960s. Although there were some promising earlystudies, many in the microbiology world believed that the capabilities of massspectrometry were overstated then, and perhaps now. The early optimism wasnot really unwarranted in that mass spectrometry studies came very close tomaking real headway in the development of rapid and reliable methods forthe characterizing of many disease-causing organisms. Two prominent lines ofmass spectrometry research illustrate this point. One technique that showedmuch promise was pyrolysis mass spectrometry (PyMS). This approach wasrapid, rugged, and seemed to provide taxonomically specific spectral profiles,especially in well-controlled laboratory settings. On this promise majorresearch efforts were put into the development of PyMS systems, especiallyfor battlefield detection of microbes used in germ warfare. Although thesesystems showed some promise for the detection of chemical agents, they wereless practical for the analysis of microorganisms in real-world settings.

It is easy to look back from todays vantage point and assert that the spec-tral patterns produced by pyrolysis were not sufficiently dissimilar for differ-ent organisms, or that pattern recognition (or computational) approaches werenot advanced enough to provide the specificity needed for routine applicationof PyMS to bacterial characterization. Such criticism misses the importantpoint that these methods did work within certain limitations. Indeed, research

xi

in PyMS and related areas continues today as investigators attempt to increasethe diversity of spectral patterns for bacteria using modifications of traditionalCurrie-point pyrolysis electron impact mass spectrometry, such as metastableatom bombardment and advanced methods for assembling libraries of pyrol-ysis data. Another approach that very nearly succeeded was the application oflaser desorption ionization mass spectrometry to bacteria. Early pioneers inthe field were attempting whole-cell studies using electron impact and laserdesorption ionization, prior to the advent of modern desorption ionizationtechniques. These studies are the direct linear predecessors of techniques suchas whole-cell MALDI-TOF MS. Had these investigators been working after,rather that before, the introduction of the now-common techniques developedto study proteins and other biopolymers, their work would have been as suc-cessful as todays. Unfortunately, their laser desorption studies of bacteriawere attempted without the benefit of a matrix to facilitate the interactionbetween the sample and the laser, or to promote the desorption/ionizationprocess.

Much of the very important early work in this field is documented in a land-mark ACS Symposium Series book edited by Catherine Fenselau. The editorsand authors of the present book hope that they can likewise reflect much ofthe important work conducted in the last decade. We anticipate that many in the microbiology community will be skeptical about current claims thatmass spectrometry can play an important role in the analysis of bacteria, espe-cially rapid characterization. Several chapters in this book were prepared by microbiologists. Even though they have had a hand in the development of mass spectrometry approaches to bacterial analysis, they are neverthelessmicrobiology-centered investigators, and their contributions should provideboth microbiology background to the mass spectrometry specialist and a per-spective on mass spectrometry approaches to the microbiologist. This bookincludes an introduction to basic concepts in mass spectrometry, and a briefhistory of the development of mass spectrometry methods for analysis of bac-teria. The most commonly used mass spectral method for the rapid analysis of bacteria is whole-cell MALDI time-of-flight (TOF) mass spectrometry.In addition to coverage of the widely used whole-cell MALDI-TOF MSapproach, a newer approach involving very high resolution Fourier transfor-mation mass spectrometry (FTMS) is also reported. Although many analysesare based on spectral comparisons, this book reflects the parallel developmentof proteome-based approaches to bacterial identification. By either approachit is now relatively routine to classify target bacteria down to the strain levelusing mass spectrometry. A number of taxonomically important proteins havenow been identified, many of them being ribosomal proteins. These aredetected because they are both abundant and easily detected analytes inMALDI mass spectrometry studies.

The most common criticism of all instrumental (chemical) methods forcharacterizing bacteria is the relatively large number of bacteria required. Typ-ically 100,000 bacteria or more or needed for analysis. Not withstanding that

xii PREFACE

pre-analysis amplification of bacteria is a common procedure for mostmethods (including standard methods used by regulatory agencies and hospi-tals), this remains a valid criticism, especially for those developing rapid analy-sis methods. Several investigators are working on approaches to concentratebacteria or bacterial biomarkers prior to MS analysis without resort to lengthyre-growth of cells in culture media. Another criticism of mass spectrometrymethods is the cost of instrumentation. For mass spectrometry to receive widespread adoption by microbiologists, a smaller footprint and reduced costwould be very desirable. Considerable progress has been made in both of theseareas and is reported some of the following chapters.

Electrospray (ESI) ionization mass spectrometry also plays in importantrole in bacterial characterization. Because it typically includes a chromato-graphic separation step, the approach is not considered as rapid as MALDIapproaches, which do not incorporate a separation. However, compared to thetimes needed to grow bacteria in culture prior to analysis, the time frame isnot lengthy, and the addition of chromatographic separation provides manyopportunities to increase specificity. ESI/MS has been used to characterize cellular biomarkers for metabolic, genomic, and proteomics fingerprinting ofbacteria, and these approaches are reported in two chapters.

Finally, methods developed for rapid detection of microorganisms by massspectrometry may also produce biomarker data that can be used in the designof better molecular probes. As noted above, many of the strain-differentiatingproteins have been identified, and this information can be used to infer infor-mation about differences in the corresponding genes at the strain level. Onechapter reports the use of strain-specific protein biomarkers, detected in massspectrometry studies of Vibrio strains from a human outbreak in the PacificNorthwest that eventually led to an understanding of the genomic changes andhave rendered existing gene-based probes less effective over time. This is but one example of the many mechanistic and biochemical advances that willlikely come from the development of methods for bacterial speciation. Itsimply reflects a link between the recognition of the molecular species thatdifferentate cells, and the elucidation of biochemical pathways that follows thedetermination of molecular structures.

Jackson O. Lay, Jr.Charles L. Wilkins

Fayetteville, Arkansas

PREFACE xiii