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Preface <p/> List of contributors <b>Microbial Characterisation; the Transition from Conventional Methods to Proteomics.</b> <p/> 1) CHANGING CONCEPTS IN THE CHARACTERISATION OF MICROBES AND THE INFLUENCE OF MASS SPECTROMETRY <p/> <i>Haroun Shah et al</i> <p/> 1.1 Background and early attempts to use mass spectrometry on microbes. <p/> 1.2 Characterisation of microorganisms by MALDI-TOF mass spectrometry; from initial ideas to the development of the first comprehensive database. <p/> 1.3 Characterisation of microorganisms from their intracellular/membrane bound protein profiles using affinity capture with particular reference to SELDI-TOF-MS. <p/> 1.4 Comparative analysis of proteomes of diverse strains within a species; use of 2-d fluorescence difference gel electrophoresis (dige). <p/> 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. <p/> 2) MICROBIAL PHYLOGENY AND EVOLUTION BASED ON PROTEIN SEQUENCES (THE CHANGE FROM TARGETED GENES TO PROTEINS) <p/> <i>Radhey Gupta</i> <p/> 2.1 Abstract <p/> 2.2 Microbial phylogeny: overview and key unresolved issues <p/> 2.3 New protein-based molecular markers for systematic and evolutionary studies <p/> 2.4 Molecular markers elucidating the evolutionary relationships among alpha (a)-proteobacteria <p/> 2.5 Molecular markers for the bacteroidetes-chlorobi phyla <p/> 2.6 Branching order and interrelationships among bacterial phyla <p/> 2.7 Importance of protein markers for discovering unique properties for different groups of bacteria <p/> 2.8 Concluding remarks <p/> 2.9 Acknowledgements <p/> 2.10 References <p/> <b>2: PROTEOMICS TOOLS AND BIOMARKER DISCOVERY.</b> <p/> 3) OVERVIEW OF THE PROTEOMIC TOOLS AND IT LINKS TO GENOMICS <p/> <i>Raju Misra.</i> <p/> 3.1 Protein identification <p/> 3.2 Peptide Mass Fingerprint (PMF) <p/> 3.3 Peptide Fragment Fingerprint (PFF) <p/> 3.4 Peptide sequencing <p/> 3.5 False discovery rates (FDR) <p/> 3.6 Validating protein identifications <p/> 3.7 Reference Database <p/> 3.8 Data storage <p/> 3.9 Biomarker discovery <p/> 3.10 Integrating genomics with proteomics <p/> 3.11 Reference List <p/> 4) HIGH THROUGHPUT BIOMARKER DISCOVERY IN MICROORGANISMS <p/> <i>Ming Fang</i> <p/> 4.1 MALDI vs ESI <p/> 4.2 Tandem Mass Spectrometry and Hybrid Mass Spectrometers <p/> 4.3 Fragmentation in Tandem Mass Spectrometry <p/> Proteomic Strategies for Protein Identification <p/> 1. Bottom-up Proteomics <p/> 2. Top-down Proteomics <p/> Multidimensional Protein Identification <p/> Mass Spectrometry Based Targeted Protein Quantification and Biomarker Discovery <p/> Selected Reaction Monitoring <p/> Conclusions <p/> 5) MALDI MASS SPECTROMETRY IMAGING, A NEW FRONTIER IN BIOSTRUCTURAL TECHNIQUES: APPLICATIONS IN BIOMEDICINE <i>Simona Francese and Malcolm R. Clench</i> <p/> 5.1 Introduction <p/> 5.2 Practical Aspects of MALDI-MSI <p/> 5.2 Applications <p/> 5.3 Microbial molecular investigation by MALDI TOF MS <p/> 5.4 Conclusions <p/> 5.5 References <p/> <b>3: PROTEIN SAMPLES PREPARATION TECHNIQUES</b> <p/> CONVENTIONAL APPROACHES FOR SAMPLE PREPARATION FOR LIQUID <p/> CHROMATOGRAPHY AND TWO-DIMENSIONAL GEL ELECTROPHORESIS <p/> <i>Vesela Encheva and Robert Parker</i> <p/> 6.1 Introduction <p/> 6.2 Cell lysis methods <p/> 6.3 Sample preparation for 2D GE <p/> 6.4 Fractionation strategies <p/> 6.5 Sample preparation for Liquid Chromatography coupled to mass <p/> 6.6 Conclusion <p/> 6.7 Reference list <p/> 7) ISOLATION AND PREPARATION OF SPORE PROTEINS AND SUBSEQUENT CHARACTERISATION BY ELECTROPHORESIS AND MASS SPECTROMETRY <i>Nicola Thorne, Saheer Gharbia and Haroun Shah</i> <p/> 7.1 Introduction <p/> 7.2 Experimental <p/> <i>2.1 Sporulation media</i> <p/> 7.3 Conclusion <p/> 8) CHARACTERIZATION OF BACTERIAL MEMBRANE PROTEINS USING A NOVEL COMBINATION OF A LIPID BASED PROTEIN IMMOBILIZATION TECHNIQUE WITH MASS SPECTROMETRY <p/> <i>Roger Karlsson, Darren Chooneea, Elisabet Carlsohn, Vesela Encheva and Haroun Shah</i> <p/> 8.1 Introduction <p/> 8.2 The surface proteome <p/> 8.3 Proteomics of pathogenic bacteria <p/> 8.4 Lipid-based protein immobilization technology <p/> 8.5 Salmonella Typhimurium – disease mechanism and outer membrane proteins <p/> 8.6 Outer membrane proteins of S. Typhimurium <p/> 8.7 Helicobacter pylori – disease mechanism and outer membrane proteins <p/> 8.8 Surface proteins of intact Helicobacter pylori <p/> 9) Wider Protein Detection from Biological Extracts by the Reduction of Dynamic Concentration Range. <p/> <i>Luc Guerrier, Egisto Boschetti and Piergiorgi Roghetti</i> <p/> 9.1 Introduction <p/> 9.2 Dealing with low-abundance protein discovery <p/> 9.3 Conclusions and future prospects <p/> 9.4 References <p/> 10) 3D-gel electrophoresis - a new development in protein analysis. <p/> <i>Robert Ventzki and Josef Stegemann</i> <p/> 10.1. Introduction <p/> 10.2. Methods <p/> 10.3 Results and discussion <p/> 10.4 References <p/> <b>SECTION 4: CHARACTERISATION OF MICROORGANISMS BY PATTERN MATCHING OF MASS SPECTRAL PROFILES AND BIOMARKER APPROACHES REQUIRING MINIMAL SAMPLE PREPARATION.</b> <p/> 11) Microbial Disease Biomarkers using ProteinChip Arrays <p/> <i>Shea Hamilton, Michael Levin, J. Simon Kroll, Paul R. Langford</i> <p/> 11.1 Introduction <p/> 11.2 Biomarker studies involving patients infected with viruses <p/> 11.3 Biomarker studies involving patients infected with parasites <p/> 11.4 Biomarker studies involving patients infected with bacteria <p/> 11.5 Other diseases of possible infectious origin <p/> 11.6 Conclusions <p/> 11.7 References <p/> <u>12) MALDI-TOF MS and microbial identification: years of experimental</u> <p/> <u>development to an established protocol.</u> <p/> <i><u>Wibke Kallow, Marcel Erhard,</u></i> <p/> <i><u>Haroun N. Shah, Emmanuel Raptakis, Martin Welker.</u></i> <p/> 12.1 Identification of Microorganisms in Clinical Routine <p/> 12.2 Mass Spectrometry and Microbiology <p/> 12.3 Mass Spectral ‘Fingerprints’ of Whole Cells <p/> 12.4 Reproducibility of Mass Spectral Fingerprints <p/> 12.5 Species and Strain Discrimination by Mass Spectrometry <p/> 12.6 Pattern Matching Approaches for automated Identification <p/> 12.7 Mass Spectral Identification of Microorganism – Requirements for Routine Diagnostics <p/> 12.8 Automated Mass Spectral Analysis of Microorganisms in Clinical Routine Diagnostics <p/> 12.9 Acknowledgements and references <p/> <b>5: Targeted Molecules and Analysis of Specific Microorganisms.</b> <p/> 13) Whole Cell MALDI Mass Spectrometry for the Rapid Characterisation of <p/> Bacteria; A Survey of Applications to Major Phyletic Lines in Microbial <p/> Kingdom. <p/> <i>Ben van Baar</i> <p/> 13.1 Introduction <p/> 13.2<a name="_Toc95220460"></a><a name="_Toc94447738">Scope</a> <p/> 13.3 <a name="_Toc95220461"></a><a name="_Toc94447739">Reproducibility</a><a name="_Toc95220462"></a><a name="_Toc94447740"></a> <p/> 13.3.1 <i>Factors concerning the sample</i> <p/> 13.4 <a name="_Toc95220463"></a><a name="_Toc94447741">Factors concerning the MALDI MS process</a><a name="_Toc94447742"></a> <p/> 13.5 Sample application and ionisation <p/> 13.5 <a name="_Toc94447743">Data analysis</a><a name="_Toc94447744"></a> <p/> 13.6 Spectrum libraries <p/> 13.6<a name="_Toc95220464"></a><a name="_Toc94447745">Whole cell MALDI MS of particular bacteria genera and species</a><a name="_Toc95220465"></a><a name="_Toc94447746"></a> <p/> <i>Bacillus</i> spp<i>.</i><a name="_Toc95220466"></a><a name="_Toc94447747"></a> <p/> <i>Staphylococcus</i> spp.<a name="_Toc95220467"></a><a name="_Toc94447748"></a> <p/> <i>Streptococcus</i> spp.<a name="_Toc95220468"></a><a name="_Toc94447750"></a> <p/> <i>Mycobacterium</i> spp.<a name="_Toc95220469"></a> <p/> Other Gram-positive bacteria<a name="_Toc95220470"></a> <p/> <i>Escherichia coli</i><a name="_Toc95220471"></a><a name="_Toc94447752"></a> <p/> Gram-negative food- and waterborne pathogen proteobacteria, other than E. Coli<a name="_Toc95220472"></a><a name="_Toc94447754"></a> <p/> Typical sexually transmitted pathogens: Neisseria <i>spp. and</i> Haemophilus <i>spp.</i><a name="_Toc95220473"></a><a name="_Toc94447755"></a> <p/> Gram-negative biothreat agent bacteria<a name="_Toc95220474"></a><a name="_Toc94447756"></a> <p/> Other Gram-negative bacteria<a name="_Toc95220475"></a><a name="_Toc94447757"></a> <p/> <i>Pathogenic Cyanobacteria</i><a name="_Toc95220476"></a><a name="_Toc94447758"></a> <p/> Strategies for the identification of biomarkers in whole cell MALDI MS spectra<a name="_Toc95220477"></a><a name="_Toc94447759"></a> <p/> Protein database consideration<a name="_Toc95220478"></a><a name="_Toc94447760"></a> <p/> On-target treatment and analysis <p/> Off-target’ Analysis and correlation with proteomics studies<a name="_Toc95220480"></a><a name="_Toc94447762"></a> <p/> General consideration of biomarker identification strategies<a name="_Toc95220481"></a><a name="_Toc94447763"></a> <p/> Conclusions and outlook <p/> 14) The power of Gel-based proteomics to understand <p/> physiology in <i>Bacillus subtilis</i> <p/> <i>Haike Antelmann and Michael Hecker</i> <p/> Introduction <p/> Results <p/> 1 Proteomics of protein secretion mechanisms in <i>Bacillus subtilis</i> <p/> 1.1. Protein export machineries of <i>B. subtilis</i> <p/> 1.1 The extracellular proteome of <i>B. subtilis</i> <p/> 1.2 The cell wall proteome of <i>B. subtilis</i> <p/> 1.3. The membrane attached lipoproteome of <i>B. subtilis</i> <p/> 1.3 The proteome analysis of protein secretion mechanisms in <i>B. subtilis</i> <p/> <u>2 Definition of proteomic signatures to study cell physiology</u> <p/> <u>2.1. Proteomic signatures of <i>B. subtilis</i> in response to stress and starvation</u> <p/> <u>2.2. Proteomic signatures of <i>B. subtilis</i> in response to thiol-reactive electrophiles uncovered novel regulatory mechanisms</u> <p/> 2.3. The MarR/DUF24-family YodB repressor is directly sensing thiol- reactive electrophiles <i>via</i> the conserved Cys6 residue <p/> 3 Proteomics as tool to visualize reversible and irreversible thiol- modifications <p/> <u>3.1. The thiol-redox proteome of <i>B. subtilis</i> in response to diamide and quinones</u> <p/> <u>3.2. Depletion of thiol-containing proteins by quinones due to thiol-(S)- alkylation</u> <p/> <u>4 Proteomics as tool to define regulon structures and targets for non- coding RNAs</u> <p/> <u>5 Acknowledgment</u> <p/> 15) Mass Spectrometry in the study of Tularemia Pathogenesis. <p/> <i>Jiri Stulik, Juraj Lenco, Jiri Dresler, Jana Klimentova, Lenka Hernychova, Lucie Balonova and Alena Fucikova.</i> <p/> <i>15.1</i> Introduction to molecular p<st1:PersonName w:st="on">at</st1:PersonName>hogenesis of <i>Francisella tularensis</i> infection <p/> <i>15.2 Francisella tularensis</i> LVS proteome alter<st1:PersonName w:st="on">at</st1:PersonName>ions induced by different temper<st1:PersonName w:st="on">at</st1:PersonName>ures and st<st1:PersonName w:st="on">at</st1:PersonName>ionary phase of growth <p/> 15.3Analysis of membrane protein complexes of <i>Francisella tularensis</i> <p/> 15.4 Analysis of <i>Francisella tularensis</i> glycoproteins and phosphoproteins <p/> 15.5Identific<st1:PersonName w:st="on">at</st1:PersonName>ion of <i>Francisella tularensis</i> transcription factors potentially involved in its virulence <p/> 15.6 Acknowledgements <p/> References <p/> 16) Bacterial Post-Genomics for Vaccine development <p/> <i>Giulia Bernardini, Daniela Braconi and Annalisa Santucci</i> <p/> Summary <p/> comparative genomics <p/> transcriptomics <p/> proteomics and immmunoproteomics <p/> other high-throughput technologies <p/> meningococcal vaccines and reverse vaccinology <p/> <i>helicobacter pylori</i> vaccines <p/> conclusions <p/> references <p/> <b>6 Statistical Analysis of 2D Gels and Analysis of Mass Spectral Data</b> <ol> <li/>Machine Learning Techniques for the Analysis of Mass spectrometry Data. </ol> <p/> <i>Graham Ball and Ali Al-Shahib</i> <p/> 17.1 Introduction <p/> 17.2 Pre-processing MS data <p/> 17.3 Classification of MS data <p/> 17.4 Evaluation of Classification Models <p/> 18) Mass Spectrometry for microbial Proteomics: Issues in data analysis with <p/> electrophoretic or mass spectrometric expression proteomic data. <p/> <i>Natasha A. Karp</i> <p/> Title page <p/> Foreword <p/> 18.1 Introduction <p/> 18.2 Experimental design <p/> 18.3 Data analysis <p/> 18.4 Validation <p/> 18.5 Conclusions <p/> 18.6 Figure legends <p/> 18.7 References <p/> <b>Section 7: DNA Resequencing by MALDI-TOF-Mass Spectrometry and its</b> <p/> <b>Application to Traditional Microbiological Problems.</b> <p/> (19) Comparative DNA sequence analysis and typing using Mass <p/> Spectrometry <p/> <i>Christiane Honisch,Yong Chen and Franz Hillenkamp</i> <p/> 19.1 Introduction <p/> 19.2 Comparative Sequence Analysis by MALDI-TOF MS <p/> 19.3 Applications of nucleic acid analysis by MALDI-TOF MS in clinical microbiology <p/> 19.4 Conclusion <p/> References <p/> (20) Transfer of a Traditional Serotyping System (Kauffmann-White) <p/> onto a MALDI-TOF-MS platform for the rapid Typing of <i>Salmonella</i> <p/> isolates<i>.</i> <p/> <i>Chloe Bishop, Cath Arnold and Saheer Gharbia</i> <p/> Typing of salmonella isolates <p/> <b>1.1 Introduction</b> <p/> <b>1.2 Salmonella, the pathogen</b> <p/> Biology <p/> Pathogenesis <p/> Clinical Disease <p/> <b>1.3 Complex genetic structure and the need to subtype this genus</b> <p/> Phylogeny <p/> Virulence and Gene Transfer <p/> Necessity to subtype <p/> ><b>1.4 Antigenic Analysis - The Traditional Kauffmann - White Schema and its future</b> <p/> Serotyping <p/> Flagellar Antigens <p/> Flagellar Variation <p/> Somatic Antigens <p/> <b>1.5 Sequence-based methods to determine serotypes</b> <p/> Flagellin sequences correspond directly to Salmonella serotype. <p/> Specific SNPs <p/> Subtyping by antigen sequence <p/> Variation of the <i>Rfb</i> Genes <p/> <b>1.6 Transferring the Sequences to a MALDI platform for Rapid Analysis</b> <p/> Intro <p/> Different methods available <p/> MALDI-TOF data analysis <p/> <i>Salmonella</i> molecular serotyping as a Case Study <p/> Gene Selection <p/> Results Overview <p/> Clustering and Sequence Variation of Amplicons <p/> <b>1.7 Conclusions and Summary</b> <p/> Closing Remarks

<p>“Summary Mass Spectrometry of Microbial Proteomics provides an authoritative guide to the expanding field of microbial proteomics.”  (<i>Anal Bioanal Chem</i>, 2011)</p> <p> </p>

<p><strong>Professor Haroun N. Shah</strong> 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. <p><strong>Professor Saheer E. Gharbia</strong> 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.

New advances in proteomics, driven largely by developments in mass spectrometry, continue to reveal the complexity and diversity of pathogenic mechanisms among microbes that underpin infectious diseases. Therefore a new era in medical microbiology is demanding a rapid transition from current procedures to high throughput analytical systems for the diagnosis of microbial pathogens.<br /> <p><br /> </p> <p>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.</p>

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