BSL3 and BSL4 Agents: Proteomics, Glycomics, and Antigenicity -- Contents -- Preface -- List of Contributors -- 1: Introduction: Application of Proteomic Technologies for the Analysis of Microbial Infections -- 1.1 Introduction -- 1.2 Search for New Factors of Virulence and Potential Diagnostic Markers -- 1.3 Search for New Vaccine Candidates -- 1.4 Analysis of Post-Translational Modifications of Bacterial Proteins and Protein-Protein Interactions -- 1.5 Conclusions -- References -- Part One: Basic Proteomic Methods -- 2: Separation of Proteins and Peptides -- 2.1 Introduction -- 2.1.1 Gel-Based Separation -- 2.1.1.1 One-Dimensional Electrophoresis -- 2.1.1.2 Two-Dimensional Electrophoresis -- 2.1.1.3 Protein Staining and Image Analysis -- 2.1.1.4 2-DE Limitations -- 2.1.2 In Solution-"Gel Free" Proteomics -- 2.1.3 Column Chromatography -- 2.1.3.1 Size Exclusion Chromatography -- 2.1.3.2 Reversed-Phase Liquid Chromatography -- 2.1.3.3 Hydrophilic Interaction Liquid Chromatography -- 2.1.3.4 Ion Exchanger Chromatography -- 2.1.3.5 Affinity Chromatography -- 2.1.3.6 Multidimensional Chromatography -- 2.1.4 Liquid Phase IEF and Electrophoresis -- 2.1.5 Alternative Separation Technologies -- Acknowledgment -- References -- 3: Basic Mass Spectrometric Approaches -- 3.1 Introduction -- 3.2 Ionization -- 3.2.1 Matrix-Assisted Laser Desorption/Ionization -- 3.2.2 Electrospray Ionization -- 3.3 Mass Analyzers -- 3.3.1 Time of Flight -- 3.3.2 Reflectron TOF -- 3.3.3 Quadrupole and Ion Trap -- 3.3.4 Fourier Transformation Ion Cyclotron -- 3.3.5 Tandem Mass Analyzers -- 3.3.6 Ion Detection -- 3.4 Protein Identification -- 3.4.1 Combination of 2-DE and MS -- 3.4.2 Peptide Mass Fingerprinting -- 3.4.3 Peptide Sequencing (PMF) -- 3.4.4 Shotgun Proteomics -- 3.5 Conclusion -- Acknowledgments -- References -- 4: Quantitative Mass Spectrometric Approaches.

4.1 Introduction -- 4.1.1 Gel-Based Quantitative Proteomic Methods -- 4.1.2 Shotgun Quantitative Proteomic Methods -- 4.1.3 Labeling Methods -- 4.1.3.1 Metabolic Incorporation of Stable Isotopes -- 4.1.3.2 Enzymatic Incorporation of Stable Isotopes -- 4.1.3.3 Chemical Incorporation of Stable Isotopes -- 4.2 iTRAQ Analysis of Bacterial Pathogens -- 4.2.1 Bacterial Cell Disruption and Protein Extraction -- 4.2.2 Determination of Protein Concentration -- 4.2.3 Protein Digestion -- 4.2.4 Peptide Labeling with iTRAQ Tags -- 4.2.5 Protocol for iTRAQ Analysis of Bacterial Proteins -- References -- 5: BN-PAGE of Microbial Protein Complexes -- 5.1 Introduction -- 5.2 Methods for Studying Protein-Protein Interactions -- 5.3 Blue Native Polyacrylamide Gel Electophoresis -- 5.3.1 Sample Preparation -- 5.3.1.1 Non-Denaturing Conditions -- 5.3.1.2 Selection of Detergent and Its Optimal Concentration -- 5.3.1.3 Membrane and Cytosolic Fraction Separation -- 5.3.2 1D BN-PAGE -- 5.3.3 2D BN/SDS-PAGE -- 5.4 Evaluation of BN-PAGE-Staining, MS, Western Blotting -- 5.4.1 Staining -- 5.4.1.1 Silver Staining -- 5.4.1.2 Fluorescent Staining -- 5.4.1.3 Coomassie Staining -- 5.4.2 Mass Spectrometry -- 5.4.3 Western Blotting -- 5.4.4 Other Methods of Visualization -- 5.5 BN/SDS-PAGE of ATP Synthase of Francisella tularensis -- 5.6 Conclusion -- Acknowledgment -- References -- 6: Analysis of Francisella tularensis Glycoproteins -- 6.1 Introduction to Post-Translational Modifications in Prokaryotes -- 6.2 Methodology -- 6.2.1 Analysis of Glycosylation -- 6.2.1.1 Glycoproteomics -- 6.2.1.2 Glycomics -- 6.3 Bioinformatics -- 6.4 Application of Glycoproteomic Approach Utilizing ProQ-Emerald and DIG Glycan Kits to Francisella tularensis (F. tularensis) -- 6.4.1 Bacterial Cultures and Sample Preparation -- 6.4.1.1 Preparation of Whole-Cell Lysates.

6.4.1.2 Preparation of Membrane-Enriched Fractions -- 6.4.2 Analysis of Glycoproteins in Fractions Enriched in Membrane Proteins -- 6.4.2.1 Mini Two-Dimensional Gel Electrophoresis -- 6.4.2.2 Glycoprotein Detection Using DIG Glycan Differentiation Kit -- 6.4.2.3 Glycoprotein Detection Using Pro-Q Emerald 300 Glycoprotein Stain Kit -- 6.4.2.4 Glycoprotein Identification by Mass Spectrometry -- 6.5 Results -- 6.5.1 Glycoprotein Detection Using DIG Glycan Differentiation Kit -- 6.5.2 Glycoprotein Detection Using Pro-Q Emerald 300 Glycoprotein Stain Kit -- 6.6 Conclusion -- Acknowledgments -- References -- Part Two: Identification of Proteins and Glycans from Microorganisms as Candidate Molecules for Use in Detection/Diagnosis, Therapy, and Prophylaxis -- 7: Comparative Proteome Analysis of Strains with Differential Virulence -- 7.1 Introduction -- 7.2 Methods -- 7.3 Results -- 7.3.1 Whole-Cell Lysates -- 7.3.2 Membrane-Associated Proteins -- 7.4 Discussion -- References -- 8: Analysis of Francisella tularensis Acetonitrile Extracts -- 8.1 Introduction -- 8.2 Material and Methods -- 8.2.1 Materials -- 8.2.2 Microorganism -- 8.2.3 Preparation of Cell-Free Acetonitrile Extract -- 8.2.4 Enzymatic Digestion -- 8.2.5 MALDI-TOF MS -- 8.2.6 LC-MS/MS -- 8.3 Results -- 8.3.1 MALDI-TOF MS Analysis -- 8.3.2 LC-MS/MS Analysis -- 8.4 Conclusions -- Acknowledgments -- References -- 9: Analysis of Culture Filtrate Proteins of Francisella tularensis -- 9.1 Introduction -- 9.2 Materials and Methods -- 9.3 Results -- 9.4 Discussion -- Acknowledgments -- References -- 10: Lipopolysaccharides of Coxiella burnetii : Chemical Composition and Structure, and Their Role in Diagnosis of Q Fever -- 10.1 Introduction -- 10.2 Lipopolysaccharides of C. burnetii -- 10.2.1 Chemical Composition and Structure of LPS I -- 10.2.2 Chemical Composition and Structure of LPS II.

10.2.3 The Role of LPS I in Diagnosis of Q Fever -- 10.3 Conclusion -- Acknowledgments -- References -- 11: Mimivirus Possesses Anonymous and Unique Gene Products Endowed for Antigenic Properties -- 11.1 Introduction -- 11.2 Material and Methods -- 11.2.1 Sample Preparation -- 11.2.2 2D-PAGE and Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry -- 11.2.3 Immunization and Western Blot -- 11.3 Results -- 11.3.1 Proteomic Analysis of Mimivirus Particle -- 11.3.2 Antigenic Properties of ORFan-Encoded Mimivirus Proteins -- 11.3.3 Concluding Remarks -- References -- 12: Detection of Differentially Modified Pathogen Proteins by Western Blot after 2D Gel Electrophoresis and Identification by MALDI-TOF/TOF -- 12.1 Introduction -- 12.2 Materials and Methods -- 12.2.1 Protein Sample Preparation and Fluorescence Labeling for DIGE Analysis -- 12.2.2 2D Electrophoresis and Gel Imaging -- 12.2.3 2D Western Blotting -- 12.2.4 Protein Identification by MALDI-TOF/TOF Analysis of In-Gel Tryptic Digests -- 12.3 Results -- 12.3.1 2D-DIGE Analysis of Measles Virus Infected THP1 Cells -- 12.3.2 Detection of Different Protein Forms by 2D Western Blot -- 12.3.3 Monitoring Viral Replication and Cellular Response by Antibody Multiplexing -- 12.4 Discussion -- References -- 13: Composition and Structure of Lipid A of the Intracellular Bacteria Piscirickettsia salmonis and Coxiella burnetii -- 13.1 Introduction -- 13.2 Composition and Structure of Lipid A of P. salmonis -- 13.3 Composition and Structure of Lipid A of C. burnetii -- 13.4 Conclusion -- Acknowledgments -- References -- 14: Proteins of C oxiella burnetii and Analysis of Their Function -- 14.1 Introduction -- 14.2 Proteins of C. burnetii and Their Functions -- 14.3 Conclusion and Perspectives -- Acknowledgments -- References.

15: Subtype and Toxin Variant Identifi cation of BotulinumNeurotoxin Type A Using Proteomics Techniques -- 15.1 Introduction -- 15.2 Botulinum Neurotoxins -- 15.3 Differentiation of Botulinum Neurotoxins -- 15.4 Amino Acid Sequence Identification Using Proteomics -- 15.5 Extraction of BoNT from Complex Matrices -- 15.6 Identification of BoNT Serotype with Proteomics -- 15.7 Identification of BoNT/A Subtype with Proteomics -- 15.8 Identification of BoNT/A1 Strain with Proteomics -- 15.9 Conclusions -- Disclaimer -- References -- 16: Protein Microarrays for Antigen Discovery -- 16.1 Introduction -- 16.2 Microarray Assembly -- 16.3 Antibody Assays -- 16.4 Antigens and Proteomes of Viruses -- 16.5 Antigens and Proteomes of Pathogenic Bacteria -- 16.6 Conclusions -- Acknowledgment -- References -- 17: MALDI-TOF Mass Spectrometry for Rapid Identification of Highly Pathogenic Microorganisms -- 17.1 Introduction -- 17.2 Microbial Identification by MALDI-TOF Mass Spectrometry -- 17.2.1 Basic Principles of MALDI-TOF Mass Spectrometry -- 17.2.2 Preparation of Microbial Samples for MALDI-TOF MS -- 17.2.3 Spectral Data Analysis: Preprocessing, Calibration, Peak Detection, and Data Visualization -- 17.2.4 Multivariate Classification Analysis-Pattern Recognition Methods -- 17.2.5 Identification of Taxon-Specific Biomarkers -- 17.3 Inactivation of Highly Pathogenic Microorganisms for MALDI-TOF Mass Spectrometry -- 17.3.1 Time and Concentration Dependence of TFA Inactivation -- 17.3.2 Centrifugation-Reduction of the Supernatant's Cell Concentration -- 17.3.3 Sterile Filtration -- 17.3.4 Molecular and Structural Aspects of Spore Treatment by TFA -- 17.4 Identification of Important Bacterial Pathogenes Using MALDI-TOF MS -- 17.4.1 Bacillus anthracis -- 17.4.2 Burkholderia mallei/pseudomallei -- 17.4.3 Yersinia pestis -- 17.5 Concluding Remarks -- Acknowledgments.

References.