This book covers the broad microbiological applications of proteomics and mass spectrometry. It is divided into six sections that follow the general progression in which most microbiology laboratories are approaching the subject Transition, Tools, Preparation, Profiling by Patterns, Target Proteins, and Data Analysis.

Professor Haroun N. Shah is Head of Molecular Identification Services Unit at the Centre for Infections, Health Protection Agency, London. The Centre for Infections provides infectious disease surveillance and microbial identification services, co-ordinating the investigation and cause of national and uncommon outbreaks of diseases. Haroun holds several chairs at various universities and spent 25 years in academic life at the University of London.

Professor Saheer E. Gharbia is Head of the Applied and Functional Genomics Unit at the HPA Centre for Infections. She has a PhD in molecular Genetics and Biochemistry, and has held various academic positions at the University of London and McGill University, Canada.

List of contributorsMicrobial Characterisation; the Transition from Conventional Methods to Proteomics.

1) CHANGING CONCEPTS IN THE CHARACTERISATION OF MICROBES AND THE INFLUENCE OF MASS SPECTROMETRY

Haroun Shah et al

1.1 Background and early attempts to use mass spectrometry on microbes.

1.2 Characterisation of microorganisms by MALDI-TOF mass spectrometry; from initial ideas to the development of the first comprehensive database.

1.3 Characterisation of microorganisms from their intracellular/membrane bound protein profiles using affinity capture with particular reference to SELDI-TOF-MS.

1.4 Comparative analysis of proteomes of diverse strains within a species; use of 2-d fluorescence difference gel electrophoresis (dige).

1.5 Searching for low abundant and low molecular weight proteins and peptides using nanoparticles as a selective and concentration probes for MALDI-TOF-MS analysis.

2) MICROBIAL PHYLOGENY AND EVOLUTION BASED ON PROTEIN SEQUENCES (THE CHANGE FROM TARGETED GENES TO PROTEINS)

Radhey Gupta

2.1 Abstract

2.2 Microbial phylogeny: overview and key unresolved issues

2.3 New protein-based molecular markers for systematic and evolutionary studies

2.4 Molecular markers elucidating the evolutionary relationships among alpha (a)-proteobacteria

2.5 Molecular markers for the bacteroidetes-chlorobi phyla

2.6 Branching order and interrelationships among bacterial phyla

2.7 Importance of protein markers for discovering unique properties for different groups of bacteria

2.8 Concluding remarks

2.9 Acknowledgements

2.10 References

2: PROTEOMICS TOOLS AND BIOMARKER DISCOVERY.

3) OVERVIEW OF THE PROTEOMIC TOOLS AND IT LINKS TO GENOMICS

Raju Misra.

3.1 Protein identification

3.2 Peptide Mass Fingerprint (PMF)

3.3 Peptide Fragment Fingerprint (PFF)

3.4 Peptide sequencing

3.5 False discovery rates (FDR)

3.6 Validating protein identifications

3.7 Reference Database

3.8 Data storage

3.9 Biomarker discovery

3.10 Integrating genomics with proteomics

3.11 Reference List

4) HIGH THROUGHPUT BIOMARKER DISCOVERY IN MICROORGANISMS

Ming Fang

4.1 MALDI vs ESI

4.2 Tandem Mass Spectrometry and Hybrid Mass Spectrometers

4.3 Fragmentation in Tandem Mass Spectrometry

Proteomic Strategies for Protein Identification

1. Bottom-up Proteomics

2. Top-down Proteomics

Multidimensional Protein Identification

Mass Spectrometry Based Targeted Protein Quantification and Biomarker Discovery

Selected Reaction Monitoring

Conclusions

5) MALDI MASS SPECTROMETRY IMAGING, A NEW FRONTIER IN BIOSTRUCTURAL TECHNIQUES: APPLICATIONS IN BIOMEDICINESimona Francese and Malcolm R. Clench

5.1 Introduction

5.2 Practical Aspects of MALDI-MSI

5.2 Applications

5.3 Microbial molecular investigation by MALDI TOF MS

5.4 Conclusions

5.5 References

3: PROTEIN SAMPLES PREPARATION TECHNIQUES

CONVENTIONAL APPROACHES FOR SAMPLE PREPARATION FOR LIQUID

CHROMATOGRAPHY AND TWO-DIMENSIONAL GEL ELECTROPHORESIS

Vesela Encheva and Robert Parker

6.1 Introduction

6.2 Cell lysis methods

6.3 Sample preparation for 2D GE

6.4 Fractionation strategies

6.5 Sample preparation for Liquid Chromatography coupled to mass

6.6 Conclusion

6.7 Reference list

7) ISOLATION AND PREPARATION OF SPORE PROTEINS AND SUBSEQUENT CHARACTERISATION BY ELECTROPHORESIS AND MASS SPECTROMETRYNicola Thorne, Saheer Gharbia and Haroun Shah

7.1 Introduction

7.2 Experimental

2.1 Sporulation media

7.3 Conclusion

8) CHARACTERIZATION OF BACTERIAL MEMBRANE PROTEINS USING A NOVEL COMBINATION OF A LIPID BASED PROTEIN IMMOBILIZATION TECHNIQUE WITH MASS SPECTROMETRY

Roger Karlsson, Darren Chooneea, Elisabet Carlsohn, Vesela Encheva and Haroun Shah

8.1 Introduction

8.2 The surface proteome

8.3 Proteomics of pathogenic bacteria

8.4 Lipid-based protein immobilization technology

8.5 Salmonella Typhimurium disease mechanism and outer membrane proteins

8.6 Outer membrane proteins of S. Typhimurium

8.7 Helicobacter pylori disease mechanism and outer membrane proteins

8.8 Surface proteins of intact Helicobacter pylori

9) Wider Protein Detection from Biological Extracts by the Reduction of Dynamic Concentration Range.

Luc Guerrier, Egisto Boschetti and Piergiorgi Roghetti

9.1 Introduction

9.2 Dealing with low-abundance protein discovery

9.3 Conclusions and future prospects

9.4 References

10) 3D-gel electrophoresis - a new development in protein analysis.

Robert Ventzki and Josef Stegemann

10.1. Introduction

10.2. Methods

10.3 Results and discussion

10.4 References

SECTION 4: CHARACTERISATION OF MICROORGANISMS BY PATTERN MATCHING OF MASS SPECTRAL PROFILES AND BIOMARKER APPROACHES REQUIRING MINIMAL SAMPLE PREPARATION.

11) Microbial Disease Biomarkers using ProteinChip Arrays

Shea Hamilton, Michael Levin, J. Simon Kroll, Paul R. Langford

11.1 Introduction

11.2 Biomarker studies involving patients infected with viruses

11.3 Biomarker studies involving patients infected with parasites

11.4 Biomarker studies involving patients infected with bacteria

11.5 Other diseases of possible infectious origin

11.6 Conclusions

11.7 References

12) MALDI-TOF MS and microbial identification: years of experimental

development to an established protocol.

Wibke Kallow, Marcel Erhard,

Haroun N. Shah, Emmanuel Raptakis, Martin Welker.

12.1 Identification of Microorganisms in Clinical Routine

12.2 Mass Spectrometry and Microbiology

12.3 Mass Spectral Fingerprints of Whole Cells

12.4 Reproducibility of Mass Spectral Fingerprints

12.5 Species and Strain Discrimination by Mass Spectrometry

12.6 Pattern Matching Approaches for automated Identification

12.7 Mass Spectral Identification of Microorganism Requirements for Routine Diagnostics

12.8 Automated Mass Spectral Analysis of Microorganisms in Clinical Routine Diagnostics

12.9 Acknowledgements and references

5: Targeted Molecules and Analysis of Specific Microorganisms.

13) Whole Cell MALDI Mass Spectrometry for the Rapid Characterisation of

Bacteria; A Survey of Applications to Major Phyletic Lines in Microbial

Kingdom.

Ben van Baar

13.1 Introduction

13.2Scope

13.3Reproducibility

13.3.1Factors concerning the sample

13.4Factors concerning the MALDI MS process

13.5 Sample application and ionisation

13.5Data analysis

13.6 Spectrum libraries

13.6Whole cell MALDI MS of particular bacteria genera and species

Bacillus spp.

Staphylococcus spp.

Streptococcus spp.

Mycobacterium spp.

Other Gram-positive bacteria

Escherichia coli

Gram-negative food- and waterborne pathogen proteobacteria, other than E. Coli

Typical sexually transmitted pathogens: Neisseriaspp. and Haemophilusspp.

Gram-negative biothreat agent bacteria

Other Gram-negative bacteria

Pathogenic Cyanobacteria

Strategies for the identification of biomarkers in whole cell MALDI MS spectra

Protein database consideration

On-target treatment and analysis

Off-target Analysis and correlation with proteomics studies

General consideration of biomarker identification strategies

Conclusions and outlook

14) The power of Gel-based proteomics to understand

physiology inBacillus subtilis

Haike Antelmann and Michael Hecker

Introduction

Results

1 Proteomics of protein secretion mechanisms inBacillus subtilis

1.1. Protein export machineries ofB. subtilis

1.1 The extracellular proteome ofB. subtilis

1.2 The cell wall proteome ofB. subtilis

1.3. The membrane attached lipoproteome ofB. subtilis

1.3 The proteome analysis of protein secretion mechanisms inB. subtilis

2 Definition of proteomic signatures to study cell physiology

2.1. Proteomic signatures ofB. subtilis in response to stress and starvation

2.2. Proteomic signatures ofB. subtilis in response to thiol-reactive electrophiles uncovered novel regulatory mechanisms

2.3. The MarR/DUF24-family YodB repressor is directly sensing thiol- reactive electrophilesvia the conserved Cys6 residue

3 Proteomics as tool to visualize reversible and irreversible thiol- modifications

3.1. The thiol-redox proteome ofB. subtilis in response to diamide and quinones

3.2. Depletion of thiol-containing proteins by quinones due to thiol-(S)- alkylation

4 Proteomics as tool to define regulon structures and targets for non- coding RNAs

5 Acknowledgment

15) Mass Spectrometry in the study of Tularemia Pathogenesis.

Jiri Stulik, Juraj Lenco, Jiri Dresler, Jana Klimentova, Lenka Hernychova, Lucie Balonova and Alena Fucikova.

15.1 Introduction to molecular pathogenesis ofFrancisella tularensis infection

15.2 Francisella tularensis LVS proteome alterations induced by different temperatures and stationary phase of growth

15.3Analysis of membrane protein complexes ofFrancisella tularensis

15.4 Analysis ofFrancisella tularensis glycoproteins and phosphoproteins

15.5Identification ofFrancisella tularensis transcription factors potentially involved in its virulence

15.6 Acknowledgements

References

16) Bacterial Post-Genomics for Vaccine development

Giulia Bernardini, Daniela Braconi and Annalisa Santucci

Summary

comparative genomics

transcriptomics

proteomics and immmunoproteomics

other high-throughput technologies

meningococcal vaccines and reverse vaccinology

helicobacter pylori vaccines

conclusions

references

6 Statistical Analysis of 2D Gels and Analysis of Mass Spectral DataMachine Learning Techniques for the Analysis of Mass spectrometry Data.

Graham Ball and Ali Al-Shahib

17.1 Introduction

17.2 Pre-processing MS data

17.3 Classification of MS data

17.4 Evaluation of Classification Models

18) Mass Spectrometry for microbial Proteomics: Issues in data analysis with

electrophoretic or mass spectrometric expression proteomic data.

Natasha A. Karp

Title page

Foreword

18.1 Introduction

18.2 Experimental design

18.3 Data analysis

18.4 Validation

18.5 Conclusions

18.6 Figure legends

18.7 References

Section 7: DNA Resequencing by MALDI-TOF-Mass Spectrometry and its

Application to Traditional Microbiological Problems.

(19) Comparative DNA sequence analysis and typing using Mass

Spectrometry

Christiane Honisch,Yong Chen and Franz Hillenkamp

19.1 Introduction

19.2 Comparative Sequence Analysis by MALDI-TOF MS

19.3 Applications of nucleic acid analysis by MALDI-TOF MS in clinical microbiology

19.4 Conclusion

References

(20) Transfer of a Traditional Serotyping System (Kauffmann-White)

onto a MALDI-TOF-MS platform for the rapid Typing ofSalmonella

isolates.

Chloe Bishop, Cath Arnold and Saheer Gharbia

Typing of salmonella isolates

1.1 Introduction

1.2 Salmonella, the pathogen

Biology

Pathogenesis

Clinical Disease

1.3 Complex genetic structure and the need to subtype this genus

Phylogeny

Virulence and Gene Transfer

Necessity to subtype

>1.4 Antigenic Analysis - The Traditional Kauffmann - White Schema and its future

Serotyping

Flagellar Antigens

Flagellar Variation

Somatic Antigens

1.5 Sequence-based methods to determine serotypes

Flagellin sequences correspond directly to Salmonella serotype.

Specific SNPs

Subtyping by antigen sequence

Variation of theRfb Genes

1.6 Transferring the Sequences to a MALDI platform for Rapid Analysis

Intro

Different methods available

MALDI-TOF data analysis

Salmonella molecular serotyping as a Case Study

Gene Selection

Results Overview

Clustering and Sequence Variation of Amplicons

1.7 Conclusions and Summary

Closing Remarks