Mass Spectrometry for Microbial Proteomics -- Contents -- Preface -- List of Contributors -- Part I: Microbial Characterisation -- the Transition from Conventional Methods to Proteomics -- 1: Changing Concepts in the Characterisation of Microbes and the Influence of Mass Spectrometry -- 1.1 Background and Early Attempts to Use Mass Spectrometry on Microbes -- 1.2 Characterisation of Microorganisms by MALDI-TOF-MS -- 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 Surface-Enhanced Laser Desorption/Ionisation (SELDI)-TOF-MS -- 1.3.1 A Protein Fingerprinting Platform to Replace SDS-PAGE -- 1.3.2 A Species-Specific Diagnostic Method -- 1.3.3 A Biomarker Search Tool -- 1.4 Comparative Analysis of Proteomes of Diverse Strains within a Species -- Use of 2D Fluorescence Difference Gel Electrophoresis (DIGE) -- 1.4.1 2D GE -- 1.4.2 The DIGE Technique -- 1.5 Nanoparticles as an Alternative Approach in the Analysis and Detection of Low Abundance and Low Molecular Weight Proteins Using MALDI-TOF-MS -- References -- 2: Microbial Phylogeny and Evolution Based on Protein Sequences (The Change from Targeted Genes to Proteins) -- 2.1 Introduction -- 2.2 Bacterial 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 α-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 -- Acknowledgements -- References.

Part II: Proteomics Tools and Biomarker Discovery -- 3: Overview of Proteomic Tools and Their Links to Genomics -- 3.1 Introduction -- 3.2 Protein Identification -- 3.2.1 Peptide Mass Fingerprint (PMF) -- 3.2.2 Peptide Fragment Fingerprint -- 3.2.3 Peptide Sequencing -- 3.2.4 False Discovery Rate (FDR) -- 3.2.5 Validating Protein Identifications -- 3.2.6 Reference Database -- 3.2.7 Data Storage -- 3.3 Applications -- 3.3.1 Biomarker Discovery -- 3.3.2 Integrating Genomics with Proteomics -- References -- 4: Tandem Mass Spectrometry-Based Proteomics, Protein Characterisation and Biomarker Discovery in Microorganisms -- 4.1 Introduction -- 4.2 Mass Spectrometry -- 4.2.1 MALDI Versus ESI -- 4.2.2 Tandem Mass Spectrometry and Hybrid Mass Spectrometers -- 4.2.3 Fragmentation in Tandem Mass Spectrometry -- 4.3 Proteomic Strategies for Protein Identification -- 4.3.1 Bottom-Up Proteomics -- 4.3.2 Top-Down Proteomics -- 4.4 Multidimensional Protein Identification -- 4.5 Mass Spectrometry-Based Targeted Protein Quantification and Biomarker Discovery -- 4.5.1 Selected Reaction Monitoring -- 4.6 Conclusions -- References -- 5: MALDI Mass Spectrometry Imaging, a New Frontier in Biostructural Techniques: Applications in Biomedicine -- 5.1 Introduction -- 5.2 Practical Aspects of MALDI-MSI -- 5.2.1 Instrumentation for MALDI-MSI -- 5.3 Applications -- 5.3.1 Pharmaceuticals -- 5.3.2 MALDI-MSI and Medicine -- 5.3.3 Biotechnology -- 5.4 Microbial Molecular Investigation by MALDI-TOF-MS -- 5.4.1 Microbial MALDI-TOF-MSI -- 5.4.2 Microbial Proteomic Characterisation and Classification via MALDI-TOF-MS and MS/MS -- 5.5 Conclusions -- References -- Part III: Protein Samples: Preparation Techniques -- 6: Conventional Approaches for Sample Preparation for Liquid Chromatography and Two-Dimensional Gel Electrophoresis -- 6.1 Introduction -- 6.2 Cell Lysis Methods.

6.2.1 Mechanical Lysis -- 6.2.2 Chemical and Osmotic Lysis -- 6.2.3 Enzymatic Lysis -- 6.3 Sample Preparation for 2D GE -- 6.3.1 Removal of Interfering Substances -- 6.3.2 Solubilisation Strategies -- 6.3.3 Sample Preparation for Difference in Gel Electrophoresis (DIGE) -- 6.3.4 Preparation of Environmental Samples -- 6.4 Fractionation Strategies -- 6.4.1 Surface Associated Proteins -- 6.4.2 Secreted Proteins -- 6.5 Sample Preparation for Liquid Chromatography coupled with Mass Spectrometry (LC-MS) -- 6.5.1 Brief Background to Protein Identification by LC-MS -- 6.5.2 Pitfalls of Poor Sample Preparation in LC-MS of Peptides -- 6.5.3 General Sample Lysis Consideration -- 6.5.4 Crude Protein Purification -- 6.5.5 Protein Resolubilsation and In-Solution Digestion -- 6.5.6 Protein Digestion -- 6.5.7 Microscale Clean Up Prior to LC-MS -- 6.6 Conclusion -- References -- 7: Isolation and Preparation of Spore Proteins and Subsequent Characterisation by Electrophoresis and Mass Spectrometry -- 7.1 Introduction -- 7.1.1 The Model Organism: Bacillus subtilis -- 7.1.2 Sporulation -- 7.1.3 The Spore Structure -- 7.1.4 C. difficile and Disease -- 7.1.5 Bacterial Spores of Clostridia -- 7.2 Experimental -- 7.2.1 Sporulation Media -- 7.2.2 Spore Purification -- 7.2.3 Spore Protein Extraction and Solubilisation -- 7.3 Conclusion -- References -- 8: Characterization of Bacterial Membrane Proteins Using a Novel Combination of a Lipid Based Protein Immobilization Technique with Mass Spectrometry -- 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 H. pylori -- References -- 9: Wider Protein Detection from Biological Extracts by the Reduction of the Dynamic Concentration Range -- 9.1 Introduction -- 9.2 Fractionation as a Means to Decipher Proteome Complexity -- 9.2.1 Subcellular Fractionation -- 9.2.2 Protein Precipitation -- 9.2.3 Immunoprecipitation -- 9.2.4 Chromatographic Fractionation -- 9.2.5 Electrokinetic Methods in Proteome Fractionation -- 9.3 Dealing with Low Abundance Proteins -- 9.3.1 Depletion of a Few High Abundance Proteins -- 9.3.2 Narrow IPG -- 9.3.3 Reduction of the Dynamic Concentration Range with Combinatorial Ligand Libraries -- 9.4 Conclusions and Envisioned Outcome -- Acknowledgements -- References -- 10: 3D-Gel Electrophoresis - A New Development in Protein Analysis -- 10.1 Introduction -- 10.1.1 State of the Art in Protein Analysis -- 10.1.2 Innovations by 3D-Gel Electrophoresis -- 10.1.3 Concept of 3D-Gel Electrophoresis -- 10.2 Methods -- 10.2.1 The 3D-Gel Instrument -- 10.2.2 Thermal Management of the 3D-Gel -- 10.2.3 Online Detection of Laser-Induced Fluorescence -- 10.2.4 Casting of the 3D-Gel -- 10.2.5 Sample Preparation and Fluorescent Labelling -- 10.2.6 Sample Loading -- 10.2.7 Image Processing and Data Evaluation -- 10.3 Results and Discussion -- 10.3.1 Comparison of 3D-Gel with Standard Slab Gel Separation -- 10.3.2 Applications of 3D-Gel Electrophoresis -- 10.3.3 Future Developments -- Acknowledgements -- References -- Part IV: Characterisation of Microorganisms by Pattern Matching of Mass Spectral Profiles and Biomarker Approaches Requiring Minimal Sample Preparation -- 11: Microbial Disease Biomarkers Using ProteinChip Arrays -- 11.1 Introduction -- 11.2 Biomarker Studies Involving Patients Infected with Viruses -- 11.2.1 Hepatitis B and C -- 11.2.2 Severe Acute Respiratory Syndrome (SARS).

11.2.3 Human Immunodeficiency Virus (HIV) -- 11.2.4 Human T-Cell Leukaemia Virus Type-1 (HTLV-1) -- 11.2.5 BK Virus (BKV) -- 11.2.6 Cytomegalovirus (CMV) -- 11.2.7 Porcine Reproductive and Respiratory Disease Syndrome (PRRS) -- 11.3 Biomarker Studies Involving Patients Infected with Parasites -- 11.3.1 Trypanosomiasis -- 11.3.2 Fasciolosis -- 11.4 Biomarker Studies Involving Patients Infected with Bacteria -- 11.4.1 Tuberculosis -- 11.4.2 Infectious Endocarditis -- 11.4.3 Respiratory Diseases -- 11.4.4 Intra-Amniotic Infection -- 11.4.5 Bacterial Peritonitis -- 11.5 Other Diseases of Possible Infectious Origin -- 11.5.1 Kawasaki Disease -- 11.6 Conclusions -- References -- 12: MALDI-TOF MS for Microbial Identification: Years of Experimental Development to an Established Protocol -- 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 Microorganisms - Requirements for Routine Diagnostics -- 12.8 Automated Mass Spectral Analysis of Microorganisms in Clinical Routine Diagnostics -- Acknowledgements -- References -- Part V: 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 Lineages in the Microbial Kingdom -- 13.1 Introduction -- 13.2 Scope -- 13.3 Reproducibility -- 13.3.1 Factors Concerning the Sample -- 13.3.2 Factors Concerning the MALDI MS Process -- 13.4 Whole Cell MALDI MS of Particular Bacteria Genera and Species -- 13.4.1 Bacillus spp. -- 13.4.2 Staphylococcus spp. -- 13.4.3 Streptococcus spp.

13.4.4 Mycobacterium spp.