Mass Spectrometry For Microbial Proteomics

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.


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.

Preface


List of contributors Microbial 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 BIOMEDICINE Simona 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 SPECTROMETRY Nicola 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


13.3 Reproducibility


13.3.1 Factors concerning the sample


13.4 Factors concerning the MALDI MS process


13.5 Sample application and ionisation


13.5 Data 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: Neisseria spp. and Haemophilus spp.


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 in Bacillus subtilis


Haike Antelmann and Michael Hecker


Introduction


Results


1 Proteomics of protein secretion mechanisms in Bacillus subtilis


1.1. Protein export machineries of B. subtilis


1.1 The extracellular proteome of B. subtilis


1.2 The cell wall proteome of B. subtilis


1.3. The membrane attached lipoproteome of B. subtilis


1.3 The proteome analysis of protein secretion mechanisms in B. subtilis


2 Definition of proteomic signatures to study cell physiology


2.1. Proteomic signatures of B. subtilis in response to stress and starvation


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


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


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


3.1. The thiol-redox proteome of B. 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 of Francisella tularensis infection


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


15.3Analysis of membrane protein complexes of Francisella tularensis


15.4 Analysis of Francisella tularensis glycoproteins and phosphoproteins


15.5Identification of Francisella 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 Data


* Machine 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 of Salmonella


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 the Rfb 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