1 - Title Page [Seite 5]
2 - Copyright Page [Seite 6]
3 - Contents [Seite 9]
4 - List of Contributors [Seite 23]
5 - Preface A Brief Tour of the Technology and New Grounds for Innovation [Seite 31]
6 - Part I MALDI-TOF Mass Spectrometry [Seite 35]
6.1 - 1 A Paradigm Shift from Research to Front-Line Microbial Diagnostics in MALDI-TOF and LC-MS/MS: A Laboratory's Vision and Relentless Resolve to Help Develop and Implement This New Technology amidst Formidable Obstacles [Seite 37]
6.1.1 - 1.1 Introduction [Seite 37]
6.1.1.1 - 1.1.1 Personal Experience at the Interface of Systematics and Diagnostics [Seite 38]
6.1.1.2 - 1.1.2 MALDI-TOF MS: The Early Years [Seite 38]
6.1.1.3 - 1.1.3 The Formidable Challenge to Gain the Confidence of the Clinical Microbiologist in MALDI-TOF MS [Seite 40]
6.1.2 - 1.2 Overcoming the Variable Parameters of MALDI-TOF MS Analysis: Publication of the First Database in 2004 [Seite 42]
6.1.3 - 1.3 SELDI-TOF MS: A Powerful but Largely Unrecognized Microbiological MALDI-TOF MS Platform [Seite 50]
6.1.4 - 1.4 MALDI-TOF MS as a Platform for DNA Sequencing [Seite 52]
6.1.5 - 1.5 Insights into the Proteome of Major Pathogens 2005-2009: Field Testing of MALDI-TOF MS [Seite 55]
6.1.6 - 1.6 2010-2011: The Triumph of MALDI-TOF MS and Emerging Interest in Tandem MS for Clinical Microbiology [Seite 56]
6.1.7 - 1.7 Preparations for MALDI-TOF MS Analysis on a Grand Scale: The Looming London 2012 Olympics [Seite 59]
6.1.8 - 1.8 Investigating the Detection and Pathogenic Potential of E. coli O104:H4 during Outbreak of 2011 [Seite 60]
6.1.8.1 - 1.8.1 The Transition from MALDI-TOF MS to High-Resolution LC-MS/MS: Merits of Bottom-Up and Top-Down Proteomics for Microbial Characterization [Seite 63]
6.1.9 - 1.9 Conclusions [Seite 67]
6.1.10 - References [Seite 68]
6.2 - Chapter 2 Criteria for Development of MALDI-TOF Mass Spectral Database [Seite 73]
6.2.1 - 2.1 Introduction [Seite 73]
6.2.2 - 2.2 Commercially Available Databases [Seite 73]
6.2.3 - 2.3 Establishment of User?Defined Databases [Seite 75]
6.2.4 - 2.4 Species Identification/Control of Reference Strains to Be Included into a Database [Seite 76]
6.2.5 - 2.5 Sample Preparation [Seite 77]
6.2.5.1 - 2.5.1 Microorganism Cultivation [Seite 77]
6.2.5.2 - 2.5.2 MALDI Sample Preparation [Seite 78]
6.2.6 - 2.6 MALDI-TOF MS Measurement [Seite 79]
6.2.7 - 2.7 Quality Control during Creation and after Establishment of Reference Libraries [Seite 80]
6.2.8 - 2.8 Common Influencing Factors for MALDI-TOF MS [Seite 80]
6.2.8.1 - 2.8.1 Influencing Factors, Specifically Weighted for MALDI Biotyper [Seite 80]
6.2.8.2 - 2.8.2 Selection of Strains [Seite 81]
6.2.8.3 - 2.8.3 Sample Preparation for Measurement [Seite 81]
6.2.8.4 - 2.8.4 Mass Spectrometry Measurement [Seite 82]
6.2.8.5 - 2.8.5 Spectra Analysis/Quality Control [Seite 82]
6.2.8.6 - 2.8.6 MSP Creation and Analysis/Quality Control [Seite 84]
6.2.9 - 2.9 User-Created and Shared Databases: Examples and Benefits [Seite 84]
6.2.10 - References [Seite 85]
6.3 - Chapter 3 Applications of MALDI-TOF Mass Spectrometry in Clinical Diagnostic Microbiology [Seite 89]
6.3.1 - 3.1 Introduction [Seite 89]
6.3.2 - 3.2 Principle of Microorganisms Identification using MALDI-TOF MS [Seite 90]
6.3.2.1 - 3.2.1 Soft Ionization and MS Applied to Microorganisms Identification [Seite 90]
6.3.2.2 - 3.2.2 Biomarker Proteins [Seite 0]
6.3.2.3 - 3.2.3 Current Commercial MALDI-TOF MS Instruments [Seite 92]
6.3.2.4 - 3.2.4 Automated Colony Picking [Seite 93]
6.3.3 - 3.3 Factors Impacting the Accuracy of MALDI-TOF MS Identifications [Seite 93]
6.3.3.1 - 3.3.1 The Importance of the Database [Seite 93]
6.3.3.2 - 3.3.2 Quality of the Spectrum and Standardization of the Pre?analytic [Seite 94]
6.3.3.3 - 3.3.3 Limit of Detection [Seite 94]
6.3.3.4 - 3.3.4 Errors and Misidentifications [Seite 94]
6.3.3.5 - 3.3.5 Mixed Bacterial Populations [Seite 94]
6.3.3.6 - 3.3.6 Closely Related Species [Seite 95]
6.3.4 - 3.4 Identification of Microorganisms from Positive Cultures [Seite 95]
6.3.4.1 - 3.4.1 Identification from Positive Cultures on Solid Media [Seite 95]
6.3.4.2 - 3.4.2 Identification from Positive Blood Cultures [Seite 98]
6.3.5 - 3.5 Identification of Microorganisms Directly from Samples [Seite 99]
6.3.5.1 - 3.5.1 Urine [Seite 99]
6.3.5.2 - 3.5.2 Cerebrospinal Fluid [Seite 101]
6.3.6 - 3.6 Microorganisms Requiring a Specific Processing for MALDI-TOF MS Identification [Seite 102]
6.3.6.1 - 3.6.1 Nocardia and Actinomycetes [Seite 102]
6.3.6.2 - 3.6.2 Mycobacteria [Seite 102]
6.3.6.3 - 3.6.3 Yeast and Fungi [Seite 103]
6.3.7 - 3.7 Detection of Antimicrobial Resistance [Seite 104]
6.3.7.1 - 3.7.1 Carbapenemase Detection [Seite 104]
6.3.7.2 - 3.7.2 Methicillin-Resistant S. aureus [Seite 105]
6.3.7.3 - 3.7.3 Vancomycin-Resistant Enterococci [Seite 105]
6.3.8 - 3.8 Detection of Bacterial Virulence Factors [Seite 105]
6.3.9 - 3.9 Typing and Clustering [Seite 106]
6.3.9.1 - 3.9.1 MRSA Typing [Seite 106]
6.3.9.2 - 3.9.2 Enterobacteriaceae Typing [Seite 107]
6.3.9.3 - 3.9.3 Typing Mycobacterium spp. [Seite 107]
6.3.10 - 3.10 Application of MALDI-TOF MS in Clinical Virology [Seite 107]
6.3.11 - 3.11 PCR-Mass Assay [Seite 108]
6.3.11.1 - 3.11.1 Application of PCR-Mass Assay in Clinical Bacteriology [Seite 108]
6.3.11.2 - 3.11.2 Application of PCR-Mass Assay in Clinical Virology [Seite 108]
6.3.12 - 3.12 PCR-ESI MS [Seite 109]
6.3.13 - 3.13 Impact of MALDI-TOF MS in Clinical Microbiology and Infectious Disease [Seite 109]
6.3.13.1 - 3.13.1 Time to Result [Seite 109]
6.3.13.2 - 3.13.2 Impact on Patient Management [Seite 110]
6.3.13.3 - 3.13.3 Impact on Rare Pathogenic Bacteria and Difficult-to-Identify Organisms [Seite 110]
6.3.13.4 - 3.13.4 Anaerobes [Seite 111]
6.3.14 - 3.14 Identification of Protozoan Parasites [Seite 111]
6.3.15 - 3.15 Identification of Ticks and Fleas [Seite 111]
6.3.16 - 3.16 Costs [Seite 112]
6.3.17 - 3.17 Conclusions [Seite 112]
6.3.18 - References [Seite 113]
6.4 - Chapter 4 The Challenges of Identifying Mycobacterium to the Species Level using MALDI-TOF MS [Seite 127]
6.4.1 - Part 4A Modifications of Standard Bruker Biotyper Method [Seite 127]
6.4.2 - 4A.1 Taxonomic Structure of the Genus Mycobacterium [Seite 127]
6.4.3 - 4A.2 Tuberculosis-Causing Mycobacteria [Seite 129]
6.4.4 - 4A.3 Non-tuberculosis Mycobacteria [Seite 129]
6.4.5 - 4A.4 MALDI-TOF MS Mycobacteria Library and Parameters for Identification [Seite 132]
6.4.6 - 4A.5 Methods for Extraction [Seite 133]
6.4.6.1 - 4A.5.1 Method: Bruker's Protocol [Seite 133]
6.4.6.2 - 4A.5.2 The Methods of Khéchine et al., 2011 [Seite 133]
6.4.6.3 - 4A.5.3 Silica/Zirconium Bead Variation [Seite 135]
6.4.6.4 - 4A.5.4 Results and Recommendations [Seite 135]
6.4.7 - 4A.6 Protein Profiling of Cell Extracts using SELDI-TOF MS [Seite 138]
6.4.8 - 4A.7 Conclusion [Seite 138]
6.4.9 - References [Seite 140]
6.4.10 - Part 4B ASTA's MicroID System and Its MycoMp Database for Mycobacteria [Seite 144]
6.4.11 - 4B.1 Introduction [Seite 144]
6.4.11.1 - 4B.1.1 The Genus Mycobacterium, Disease and MALDI-TOF Mass Spectrometry [Seite 144]
6.4.12 - 4B.2 MycoMp Database for Mycobacterium: The ASTA Mycobacterial Database [Seite 145]
6.4.13 - 4B.3 MicroID Software [Seite 145]
6.4.14 - 4B.4 Database [Seite 146]
6.4.15 - 4B.5 MycoMP Database for Mycobacteria [Seite 147]
6.4.16 - 4B.6 Conclusion [Seite 154]
6.4.17 - References [Seite 154]
6.5 - Chapter 5 Transformation of Anaerobic Microbiology since the Arrival of MALDI-TOF Mass Spectrometry [Seite 157]
6.5.1 - 5.1 Introduction [Seite 157]
6.5.2 - 5.2 Identification in the Clinical Laboratory [Seite 159]
6.5.3 - 5.3 Pre-analytical Requirements Influence Species Identification of Anaerobic Bacteria [Seite 160]
6.5.4 - 5.4 Recent Database Developments for Anaerobes [Seite 163]
6.5.5 - 5.5 Application of the MALDI-TOF MS Method for Routine Identification of Anaerobes in the Clinical Practice [Seite 165]
6.5.6 - 5.6 The European Network for the Rapid Identification of Anaerobes (ENRIA) Project [Seite 168]
6.5.7 - 5.7 Subspecies-Level Typing of Anaerobic Bacteria Based on Differences in Mass Spectra [Seite 169]
6.5.8 - 5.8 Impact of MALDI-TOF MS on Subspecies Classification of Propionibacterium acnes: Insights into Protein Expression using ESI-MS-MS [Seite 170]
6.5.9 - 5.9 Direct Identification of Anaerobic Bacteria from Positive Blood Cultures [Seite 174]
6.5.10 - References [Seite 174]
6.6 - Chapter 6 Differentiation of Closely Related Organisms using MALDI-TOF MS [Seite 181]
6.6.1 - 6.1 Introduction [Seite 181]
6.6.2 - 6.2 Experimental Methods [Seite 183]
6.6.2.1 - 6.2.1 Strains and Traditional Identification [Seite 183]
6.6.2.2 - 6.2.2 PCR Identification [Seite 184]
6.6.2.3 - 6.2.3 MALDI-TOF MS Identification [Seite 185]
6.6.3 - 6.3 Results [Seite 187]
6.6.3.1 - 6.3.1 Semiautomated Models [Seite 187]
6.6.3.2 - 6.3.2 Automated Models [Seite 187]
6.6.3.3 - 6.3.3 Hybrid Models [Seite 189]
6.6.3.4 - 6.3.4 MALDI-TOF MS versus Traditional Identification Methods [Seite 190]
6.6.4 - 6.4 Discussion and Implications [Seite 192]
6.6.5 - Acknowledgments [Seite 196]
6.6.6 - References [Seite 196]
6.7 - Chapter 7 Identification of Species in Mixed Microbial Populations using MALDI?TOF MS [Seite 201]
6.7.1 - 7.1 Introduction [Seite 201]
6.7.2 - 7.2 A New Algorithm to Identify Mixed Species in a MALDI-TOF Mass Spectrum [Seite 202]
6.7.2.1 - 7.2.1 Mixed Spectrum Model [Seite 202]
6.7.2.2 - 7.2.2 Algorithm Description [Seite 204]
6.7.2.3 - 7.2.3 A Simulation Framework to Optimize the Model Parameters [Seite 206]
6.7.3 - 7.3 Toward Direct-Sample Polymicrobial Identification from Positive Blood Cultures [Seite 206]
6.7.3.1 - 7.3.1 Microbial Panel Considered [Seite 208]
6.7.3.2 - 7.3.2 Qualifying the Success of the Identification [Seite 208]
6.7.3.3 - 7.3.3 In Silico Experiments [Seite 209]
6.7.4 - 7.4 In Vitro Experiments [Seite 212]
6.7.5 - 7.5 Discussion and Perspectives [Seite 215]
6.7.6 - References [Seite 218]
6.8 - Chapter 8.1 Microbial DNA Analysis by MALDI-TOF Mass Spectrometry [Seite 221]
6.8.1 - Part 8A DNA Analysis of Viral Genomes using MALDI-TOF Mass Spectrometry [Seite 221]
6.8.2 - 8A.1 Introduction [Seite 221]
6.8.3 - 8A.2 The Molecular Detection and Identification of Viruses [Seite 222]
6.8.4 - 8A.3 Viral Quantification [Seite 223]
6.8.5 - 8A.4 The Characterization of Viral Genetic Heterogeneity [Seite 224]
6.8.6 - 8A.5 Viral Transmission Monitoring [Seite 226]
6.8.7 - 8A.6 Additional Nucleic Acid Applications of MALDI-TOF MS [Seite 227]
6.8.8 - 8A.7 Conclusion [Seite 227]
6.8.9 - References [Seite 227]
6.8.10 - Part 8B Mass Spectral Analysis of Proteins of Nonculture and Cultured Viruses [Seite 231]
6.8.11 - 8B.1 Introduction [Seite 231]
6.8.12 - 8B.2 Norovirus Identification using MS [Seite 233]
6.8.13 - 8B.3 Sample Preparation Considerations [Seite 234]
6.8.14 - 8B.4 Experimental Workflow [Seite 234]
6.8.15 - 8B.5 Detection of Intact VP1 using MALDI-TOF and SELDI-TOF MS [Seite 234]
6.8.16 - 8B.6 Peptide Mass Fingerprinting [Seite 236]
6.8.17 - 8B.7 Conclusions [Seite 237]
6.8.18 - 8B.8 Bacteriophage Identification using MS [Seite 240]
6.8.19 - 8B.9 Bacteriophages [Seite 240]
6.8.20 - 8B.10 Protein Identification [Seite 240]
6.8.21 - 8B.11 Conclusions [Seite 242]
6.8.22 - References [Seite 242]
6.9 - Chapter 9 Impact of MALDI-TOF MS in Clinical Mycology [Seite 245]
6.9.1 - 9.1 Introduction [Seite 245]
6.9.2 - 9.2 Evolution in Commercial Methodologies of Sample Preparation [Seite 247]
6.9.2.1 - 9.2.1 Fungal Identification [Seite 247]
6.9.2.2 - 9.2.2 MALDI Biotyper [Seite 248]
6.9.2.3 - 9.2.3 VITEK® MS [Seite 251]
6.9.2.4 - 9.2.4 MS LT2-ANDROMAS [Seite 252]
6.9.3 - 9.3 Effect of In-House Sample Preparation on Database Reliability [Seite 252]
6.9.3.1 - 9.3.1 Yeast Identification in Pure Culture [Seite 252]
6.9.3.2 - 9.3.2 Filamentous Fungi Identification [Seite 256]
6.9.4 - 9.4 Conclusion [Seite 259]
6.9.5 - References [Seite 260]
6.10 - Chapter 10 Development and Application of MALDI-TOF for Detection of Resistance Mechanisms [Seite 265]
6.10.1 - 10.1 Attempts to Correlate Signature Mass Ions in MALDI-TOF MS Profiles with Antibiotic Resistance [Seite 265]
6.10.2 - 10.2 Distribution and Spread of Carbapenems and Mass Spectrometry [Seite 267]
6.10.3 - 10.3 Carbapenem-Resistant Enterobacteriaceae [Seite 268]
6.10.4 - 10.4 MALDI-TOF MS Detection Based upon Changes in Antibiotic Structure due to Bacterial Degradation Enzymes [Seite 268]
6.10.5 - 10.5 Optimization of the Carbapenemase MALDI-TOF MS-Based Assay to Minimize the Time-to-Result [Seite 270]
6.10.6 - 10.6 Detection of Other Bacterial Enzymic Modifications to Antibiotic Structures [Seite 272]
6.10.7 - 10.7 Isotopic Detection using MALDI-TOF MS [Seite 273]
6.10.8 - 10.8 Multi-Resistant Pseudomonas aeruginosa [Seite 276]
6.10.9 - 10.9 MALDI Biotyper Antibiotic Susceptibility Test Rapid Assay (MBT-ASTRAT) [Seite 276]
6.10.10 - 10.10 The Potential Use of Mass Spectrometry for Antibiotic Testing in Yeast [Seite 278]
6.10.11 - References [Seite 279]
6.11 - Chapter 11 Discrimination of Burkholderia Species, Brucella Biovars, Francisella tularensis and Other Taxa at the Subspecies Level by MALDI-TOF Mass Spectrometry [Seite 283]
6.11.1 - 11.1 Introduction [Seite 283]
6.11.2 - 11.2 Principles of MALDI-TOF MS?Based Identification of Bacteria [Seite 283]
6.11.3 - 11.3 Generality versus Specificity [Seite 284]
6.11.4 - 11.4 Shigatoxin-Producing and Enterohemorrhagic Escherichia coli (STEC and EHEC) [Seite 285]
6.11.5 - 11.5 Francisella tularensis [Seite 287]
6.11.6 - 11.6 The Genus Brucella [Seite 289]
6.11.7 - 11.7 The Genus Burkholderia [Seite 290]
6.11.8 - 11.8 Studying Closely Related Organisms by MALDI-TOF MS [Seite 291]
6.11.8.1 - 11.8.1 Sample Selection [Seite 292]
6.11.8.2 - 11.8.2 Spectrum Processing [Seite 292]
6.11.8.3 - 11.8.3 Choosing Software for Statistical Calculations [Seite 292]
6.11.8.4 - 11.8.4 Search for Taxon-Specific Markers [Seite 293]
6.11.8.5 - 11.8.5 Spectrum-Based Cluster Analysis [Seite 293]
6.11.8.6 - 11.8.6 Statistical Models for Classification [Seite 293]
6.11.8.7 - 11.8.7 External Validation [Seite 294]
6.11.9 - 11.9 Conclusion [Seite 294]
6.11.10 - References [Seite 295]
6.12 - Chapter 12 MALDI-TOF-MS Based on Ribosomal Protein Coding in S10-spc-alpha Operons for Proteotyping [Seite 303]
6.12.1 - 12.1 Introduction [Seite 303]
6.12.2 - 12.2 S10-GERMS Method [Seite 306]
6.12.2.1 - 12.2.1 Background of Proteotyping [Seite 306]
6.12.2.2 - 12.2.2 Construction Procedures of the Working Database for MALDI-TOF MS Analysis [Seite 307]
6.12.2.3 - 12.2.3 Application of Standardized S10-GERMS Method to Bacterial Typing [Seite 311]
6.12.3 - 12.3 Conclusion: Computer-Aided Proteotyping of Bacteria Based on the S10-GERMS Method [Seite 335]
6.12.4 - References [Seite 337]
7 - Part II Tandem MS/MS-Based Approaches to Microbial Characterization [Seite 345]
7.1 - Chapter 13 Tandem Mass Spectrometry Analysis as an Approach to Delineate Genetically Related Taxa [Seite 347]
7.1.1 - Part A [Seite 347]
7.1.2 - 13.1 Introduction [Seite 347]
7.1.3 - 13.2 Methods [Seite 350]
7.1.4 - 13.3 Results [Seite 355]
7.1.4.1 - 13.3.1 16S rRNA Identification [Seite 355]
7.1.4.2 - 13.3.2 MALDI-TOF MS Identification [Seite 355]
7.1.5 - 13.4 Candidate Biomarker Discovery: Shotgun Sampling of Enterobacteriaceae Proteomes by GeLC-MS/MS [Seite 359]
7.1.5.1 - 13.4.1 Database Optimization and Testing [Seite 359]
7.1.5.2 - 13.4.2 Demonstrating Capability to Delineate Pathotypes using E. coli 0104:H4 as an Exemplar [Seite 359]
7.1.6 - 13.5 Discussion [Seite 365]
7.1.7 - Part B [Seite 367]
7.1.8 - 13.6 Highly Pathogenic Biothreat Agents [Seite 367]
7.1.9 - 13.7 Bacillus anthracis [Seite 368]
7.1.9.1 - 13.7.1 Methods: Strain Panel [Seite 369]
7.1.9.2 - 13.7.2 Whole Cell Protein Extraction [Seite 369]
7.1.9.3 - 13.7.3 One-Dimensional SDS-PAGE and In-Gel Digestion of Bacterial Proteins [Seite 370]
7.1.9.4 - 13.7.4 In-Solution Protein Digestion Directly from Protein Extracts [Seite 370]
7.1.9.5 - 13.7.5 1-D Nanoflow LC-MS/MS, Data-Dependent and Targeted MS Analysis [Seite 370]
7.1.9.6 - 13.7.6 Bioinformatic Workflow for Biomarker Detection [Seite 371]
7.1.9.7 - 13.7.7 Protein/Peptide Marker Identification [Seite 371]
7.1.9.8 - 13.7.8 Procedure for DNA Extraction [Seite 372]
7.1.9.9 - 13.7.9 DNA Extraction [Seite 372]
7.1.9.10 - 13.7.10 Genetic Validation of Candidate Peptide Biomarkers [Seite 372]
7.1.10 - 13.8 Summary of Results [Seite 376]
7.1.11 - 13.9 Yersinia pestis [Seite 378]
7.1.12 - 13.10 Method: Strain Panel [Seite 378]
7.1.12.1 - 13.10.1 Procedure for Whole Cell Protein Extraction [Seite 378]
7.1.12.2 - 13.10.2 One-Dimensional SDS-PAGE and In?Gel Digestion of Bacterial Proteins [Seite 378]
7.1.12.3 - 13.10.3 One-Dimensional Nanoflow LC-MS/MS, Data-Dependent and Targeted MS Analysis [Seite 378]
7.1.12.4 - 13.10.4 Bioinformatic Workflow for Biomarker Detection [Seite 379]
7.1.12.5 - 13.10.5 Genetic Validation of Peptide Biomarkers [Seite 379]
7.1.13 - 13.11 Summary of Results [Seite 379]
7.1.14 - 13.12 Fransicella tularensis [Seite 380]
7.1.15 - 13.13 Method [Seite 380]
7.1.15.1 - 13.13.1 Strain Panel [Seite 380]
7.1.15.2 - 13.13.2 Procedure for Whole Cell Protein Extraction [Seite 380]
7.1.15.3 - 13.13.3 One-Dimensional SDS-PAGE and In-Gel Digestion of Bacterial Proteins [Seite 381]
7.1.15.4 - 13.13.4 One-Dimensional Nanoflow LC-MS/MS, Data-Dependent and Targeted MS Analysis [Seite 381]
7.1.15.5 - 13.13.5 Bioinformatic Workflow for Biomarker Detection [Seite 381]
7.1.15.6 - 13.13.6 Genetic Validation of Peptide Biomarkers [Seite 381]
7.1.16 - 13.14 Summary of Results [Seite 382]
7.1.17 - 13.15 Clostridium botulinum [Seite 384]
7.1.18 - 13.16 Method [Seite 385]
7.1.18.1 - 13.16.1 Strain Panel [Seite 385]
7.1.18.2 - 13.16.2 Procedure for Whole Cell Protein Extraction [Seite 385]
7.1.18.3 - 13.16.3 One-Dimensional SDS-PAGE and In-Gel Digestion of Bacterial Proteins [Seite 386]
7.1.18.4 - 13.16.4 1-D Nanoflow LC-MS/MS, Data-Dependent and Targeted MS Analysis [Seite 386]
7.1.18.5 - 13.16.5 Bioinformatic Workflow for Biomarker Detection [Seite 386]
7.1.18.6 - 13.16.6 Procedure for DNA Extraction [Seite 387]
7.1.18.7 - 13.16.7 Genetic Validation of Peptide Biomarkers [Seite 387]
7.1.19 - 13.17 Summary of Results [Seite 389]
7.1.20 - 13.18 Burkholderia pseudomallei and B. mallei [Seite 389]
7.1.21 - 13.19 Method [Seite 391]
7.1.21.1 - 13.19.1 Strain Panel [Seite 391]
7.1.21.2 - 13.19.2 Procedure for Whole Cell Protein Extraction [Seite 391]
7.1.21.3 - 13.19.3 One-Dimensional SDS-PAGE and In-Gel Digestion of Bacterial Proteins [Seite 392]
7.1.21.4 - 13.19.4 One-Dimensional Nanoflow LC-MS/MS, Data-Dependent and Targeted MS Analysis [Seite 392]
7.1.21.5 - 13.19.5 Bioinformatic Workflow for Biomarker Detection [Seite 392]
7.1.21.6 - 13.19.6 Procedure for DNA Extraction [Seite 392]
7.1.21.7 - 13.19.7 Genetic Validation of Peptide Biomarkers [Seite 392]
7.1.22 - 13.20 Summary of Results [Seite 394]
7.1.23 - 13.21 Biomarker Detection Sensitivity and Quantification [Seite 395]
7.1.24 - 13.22 Method [Seite 395]
7.1.24.1 - 13.22.1 Preparation of Stable Isotope?Labelled Peptides [Seite 396]
7.1.24.2 - 13.22.2 Preparation of Samples for Absolute Quantification [Seite 396]
7.1.24.3 - 13.22.3 One-Dimensional Nanoflow LC-MS/MS, Data-Dependent MS Analysis [Seite 396]
7.1.24.4 - 13.22.4 Data Analysis [Seite 396]
7.1.25 - 13.23 Summary of Results [Seite 397]
7.1.26 - 13.24 Assay Sensitivity in Relation to Bacterial Cell Numbers [Seite 399]
7.1.26.1 - 13.24.1 Method [Seite 399]
7.1.26.2 - 13.24.2 Preparation of Cell Dilutions [Seite 399]
7.1.26.3 - 13.24.3 Cell Lysis Procedure [Seite 399]
7.1.26.4 - 13.24.4 Capture of Cells and Protein Material [Seite 401]
7.1.26.5 - 13.24.5 Trypsin Digestion on Filters [Seite 401]
7.1.27 - 13.25 Summary of Results [Seite 401]
7.1.28 - 13.26 Spiked Samples [Seite 402]
7.1.29 - 13.27 Method [Seite 402]
7.1.30 - 13.28 Summary of Results [Seite 402]
7.1.31 - 13.29 Spiked Cells [Seite 404]
7.1.32 - 13.30 Method [Seite 404]
7.1.33 - 13.31 Summary of Results [Seite 404]
7.1.34 - 13.32 B. anthracis Spore Analysis [Seite 404]
7.1.35 - 13.33 Method [Seite 404]
7.1.36 - 13.34 Summary of Results [Seite 405]
7.1.37 - 13.35 Assay Sensitivity in Relation to Bacterial Spore Numbers [Seite 405]
7.1.38 - 13.36 Method [Seite 405]
7.1.39 - 13.37 Summary of Results [Seite 406]
7.1.40 - 13.38 Summary of Results for Biomarker Detection Sensitivity [Seite 406]
7.1.41 - References [Seite 409]
7.2 - Chapter 14 Mapping of the Proteogenome of Clostridium difficile Isolates of Varying Virulence [Seite 413]
7.2.1 - 14.1 Introduction [Seite 413]
7.2.2 - 14.2 Virulence of Clostridium difficile [Seite 414]
7.2.2.1 - 14.2.1 Virulence Factors [Seite 414]
7.2.2.2 - 14.2.2 Variation between Strains [Seite 414]
7.2.3 - 14.3 Current Genomic and Proteomic Data [Seite 415]
7.2.4 - 14.4 Comparison of Strains of Varying Virulence [Seite 415]
7.2.5 - 14.5 Genomic Analysis of Clostridium difficile [Seite 416]
7.2.5.1 - 14.5.1 Using Roche's Flx and Junior [Seite 416]
7.2.5.2 - 14.5.2 PacBio Genomic Analysis [Seite 417]
7.2.6 - 14.6 Proteomic Analysis of Clostridium difficile [Seite 418]
7.2.6.1 - 14.6.1 Two-Dimensional Reference Mapping [Seite 418]
7.2.6.2 - 14.6.2 Differential In-Gel Electrophoresis (DIGE) [Seite 419]
7.2.6.3 - 14.6.3 One-Dimensional Gel Electrophoresis Coupled with LC-MS/MS [Seite 421]
7.2.7 - 14.7 Mapping the Proteogenome of Clostridium difficile to Phenotypic Profiles [Seite 422]
7.2.7.1 - 14.7.1 Toxin Expression [Seite 422]
7.2.7.2 - 14.7.2 Mucosal Adherence [Seite 423]
7.2.7.3 - 14.7.3 Flagella [Seite 424]
7.2.8 - 14.8 Antibiotic Resistance [Seite 428]
7.2.9 - 14.9 Conclusion [Seite 429]
7.2.10 - References [Seite 429]
7.3 - Chapter 15 Determination of Antimicrobial Resistance using Tandem Mass Spectrometry [Seite 433]
7.3.1 - 15.1 Antibiotic Resistance Mechanisms [Seite 433]
7.3.2 - 15.2 Detection of ?-lactamase Activity [Seite 435]
7.3.3 - 15.3 Other MALDI-TOF MS Methods [Seite 437]
7.3.4 - 15.4 Liquid Chromatography Coupled with MS [Seite 438]
7.3.5 - 15.5 Proteomics Approaches for Detection of Antibiotic Resistance [Seite 444]
7.3.6 - 15.6 Conclusion [Seite 448]
7.3.7 - References [Seite 449]
7.4 - Chapter 16 Proteotyping: Tandem Mass Spectrometry Shotgun Proteomic Characterization and Typing of Pathogenic Microorganisms [Seite 453]
7.4.1 - 16.1 Introduction [Seite 453]
7.4.2 - 16.2 MS and Proteomics [Seite 454]
7.4.3 - 16.3 MALDI TOF MS [Seite 456]
7.4.4 - 16.4 Tandem MS Shotgun Proteomic Analyses [Seite 460]
7.4.5 - 16.5 Top-Down Proteomics [Seite 460]
7.4.6 - 16.6 Bottom-Up Proteomics [Seite 462]
7.4.7 - 16.7 Proteotyping [Seite 464]
7.4.8 - 16.8 Matching MS Spectra to Peptides [Seite 468]
7.4.9 - 16.9 Mapping Peptides to Reference Sequences [Seite 469]
7.4.10 - 16.10 Taxonomic Assignment of Protein Sequences [Seite 470]
7.4.11 - 16.11 Challenges Assigning Fragments to Lower Taxonomic Levels [Seite 471]
7.4.12 - 16.12 Proteotyping for Diagnosing Infectious Diseases [Seite 473]
7.4.13 - 16.13 Outlook [Seite 475]
7.4.14 - 16.14 Conclusion [Seite 477]
7.4.15 - Acknowledgments [Seite 478]
7.4.16 - References [Seite 478]
7.5 - Chapter 17 Proteogenomics of Pseudomonas aeruginosa in Cystic Fibrosis Infections [Seite 485]
7.5.1 - 17.1 Introduction: Pseudomonas aeruginosa as a Clinically Important Pathogen [Seite 485]
7.5.2 - 17.2 CF and Pathophysiology [Seite 486]
7.5.3 - 17.3 CF Infections [Seite 486]
7.5.4 - 17.4 Biofilm Formation in P. aeruginosa [Seite 487]
7.5.5 - 17.5 Virulence of P. aeruginosa [Seite 488]
7.5.6 - 17.6 Genomics to Study Bacterial Pathogenesis [Seite 489]
7.5.7 - 17.7 Proteomics to Study Bacterial Pathogenesis [Seite 490]
7.5.8 - 17.8 Genomics of P. aeruginosa in CF Infections [Seite 491]
7.5.9 - 17.9 Interclonal Genome Diversity [Seite 492]
7.5.10 - 17.10 Intraclonal Genome Diversity [Seite 492]
7.5.11 - 17.11 Clonal Spread of P. aeruginosa in CF Patients [Seite 493]
7.5.12 - 17.12 Parallel Evolution [Seite 493]
7.5.13 - 17.13 Mutations in Early-Stage CF P. aeruginosa Isolates [Seite 494]
7.5.14 - 17.14 Mutations in Late-Stage CF P. aeruginosa Isolates [Seite 495]
7.5.15 - 17.15 Transcriptomics of P. aeruginosa in Chronic CF Infections [Seite 496]
7.5.16 - 17.16 Proteomics of P. aeruginosa in Chronic CF Infections [Seite 498]
7.5.17 - 17.17 Applications of Proteomics to P. aeruginosa Characterization [Seite 498]
7.5.18 - 17.18 Comparative Proteomic Investigation of Bis-(3?-5?)-Cyclic-Dimeric-GMP (C-Di-GMP) Regulation in P. aeruginosa [Seite 499]
7.5.19 - 17.19 Comparative Proteomics of Mucoid and Non-Mucoid P. aeruginosa Strains [Seite 500]
7.5.20 - 17.20 Proteogenomics Reveal Shifting in Iron Uptake of CF P. aeruginosa [Seite 500]
7.5.21 - 17.21 Conclusion and Future Perspectives [Seite 502]
7.5.22 - References [Seite 504]
7.6 - Chapter 18 Top-Down Proteomics in the Study of Microbial Pathogenicity [Seite 527]
7.6.1 - 18.1 Introduction [Seite 527]
7.6.2 - 18.2 Top-Down Analysis of Modified Bacterial Proteins in Targeted Mode [Seite 530]
7.6.3 - 18.3 Top-Down Analysis of Bacterial Proteins in Discovery Mode [Seite 532]
7.6.4 - 18.4 Top-Down Proteomics: The Next Step in Clinical Microbiology? [Seite 533]
7.6.5 - References [Seite 535]
7.7 - Chapter 19 Tandem Mass Spectrometry in Resolving Complex Gut Microbiota Functions [Seite 539]
7.7.1 - 19.1 Introduction [Seite 539]
7.7.1.1 - 19.1.1 Scope [Seite 539]
7.7.1.2 - 19.1.2 Strategies to Study Intestinal Microbiome [Seite 539]
7.7.2 - 19.2 MS in Microbiology [Seite 541]
7.7.3 - 19.3 Intestinal Metaproteomics Addressing All Proteins [Seite 546]
7.7.3.1 - 19.3.1 Preprocessing of the Sample [Seite 546]
7.7.3.2 - 19.3.2 Protein Extraction [Seite 547]
7.7.3.3 - 19.3.3 Protein Digestion [Seite 547]
7.7.3.4 - 19.3.4 Peptide Fractionation [Seite 547]
7.7.4 - 19.4 LC-MSMS Analysis [Seite 547]
7.7.5 - 19.5 Data Analysis [Seite 548]
7.7.5.1 - 19.5.1 Peptide Spectral Matching [Seite 548]
7.7.5.2 - 19.5.2 De Novo Sequencing [Seite 548]
7.7.5.3 - 19.5.3 Protein Quantification [Seite 549]
7.7.5.4 - 19.5.4 Metaproteomic Pipelines [Seite 549]
7.7.5.5 - 19.5.5 Data Storage [Seite 549]
7.7.6 - 19.6 Data Output and Interpretation [Seite 549]
7.7.7 - 19.7 Development of Surface Metaproteomics for Intestinal Microbiota [Seite 550]
7.7.7.1 - 19.7.1 Isolation of Bacteria from Fecal Samples [Seite 551]
7.7.7.2 - 19.7.2 Enrichment of the Surface Proteome from Fecal Bacterial Extract [Seite 551]
7.7.7.3 - 19.7.3 Detection of Surface Proteins by LC-MSMS [Seite 551]
7.7.8 - 19.8 Conclusions [Seite 556]
7.7.9 - References [Seite 557]
7.8 - Chapter 20 Proteogenomics of Non-model Microorganisms [Seite 563]
7.8.1 - 20.1 Introduction [Seite 563]
7.8.2 - 20.2 The "Proteogenomics" Concept [Seite 564]
7.8.3 - 20.3 Applications to Non-model Organisms: From Bacteria to Parasites [Seite 565]
7.8.4 - 20.4 Embracing Complexity with Metaproteogenomics [Seite 568]
7.8.5 - References [Seite 569]
7.9 - Chapter c21.A Analysis of MALDI-TOF MS Spectra using the BioNumerics Software [Seite 573]
7.9.1 - 21A.1 Introduction [Seite 573]
7.9.2 - 21A.2 Typing with MALDI-TOF MS [Seite 574]
7.9.3 - 21A.3 Preprocessing of Raw MALDI-TOF MS Data [Seite 574]
7.9.4 - 21A.4 Downsampling [Seite 575]
7.9.5 - 21A.5 Baseline Subtraction [Seite 576]
7.9.6 - 21A.6 Curve Smoothing [Seite 577]
7.9.7 - 21A.7 Peak Detection [Seite 580]
7.9.8 - 21A.8 Biological and Technical Replicates [Seite 580]
7.9.9 - 21A.9 Averaging of Replicates [Seite 583]
7.9.10 - 21A.10 Spectrum Analysis [Seite 584]
7.9.11 - 21A.11 Hierarchical Clustering [Seite 584]
7.9.12 - 21A.12 Alternatives to Cluster Analysis [Seite 588]
7.9.13 - 21A.13 Classifying Algorithms [Seite 593]
7.9.14 - 21A.14 Conclusion [Seite 595]
7.9.15 - References [Seite 595]
7.10 - Chapter c21.B Subtyping of Staphylococcus spp. Based upon MALDI-TOF MS Data Analysis [Seite 597]
7.10.1 - 21B.1 Introduction [Seite 597]
7.10.2 - 21B.2 Sample Collection [Seite 598]
7.10.3 - 21B.3 MALDI-TOF Mass Spectrometry [Seite 598]
7.10.4 - 21B.4 Cluster Analysis of Environmental Staphylococci [Seite 599]
7.10.5 - 21B.5 Antibiotic Susceptibility Test [Seite 599]
7.10.6 - 21B.6 Cluster Analysis of Staphylococcus spp. Recovered from Different Sites [Seite 600]
7.10.7 - 21B.7 Correlation of Staphylococci Recovered from Different Sites [Seite 601]
7.10.8 - 21B.8 Cluster Analysis of S. epidermidis Isolated from Different Sites [Seite 602]
7.10.9 - 21B.9 Cluster Analysis of S. aureus Isolated from Different Sites [Seite 603]
7.10.10 - 21B.10 Cluster Analysis of Staphylococcus spp. Combined with Antibiotic Susceptibility [Seite 603]
7.10.11 - 21B.11 Antibiotic Resistance Patterns of Closely Related S. epidermidis [Seite 604]
7.10.12 - 21B.12 Antibiotic Resistance Patterns of Closely Related S. aureus [Seite 604]
7.10.13 - 21B.13 Variations of Antibiotic Susceptibility of Closely Related S. epidermidis [Seite 606]
7.10.14 - 21B.14 Percentage of Multiple-Resistant Staphylococci Recovered from Each Site [Seite 606]
7.10.15 - 21B.15 Conclusion [Seite 607]
7.10.16 - References [Seite 609]
7.11 - Chapter c21.C Elucidating the Intra-Species Proteotypes of Pseudomonas aeruginosa from Cystic Fibrosis [Seite 613]
7.11.1 - 21C.1 The Emergence of Pseudomonas aeruginosa as Key Component of the Cystic Fibrosis Lung Flora [Seite 613]
7.11.2 - 21C.2 Diversity and Rational for Proteotyping [Seite 614]
7.11.3 - 21C.3 Selecting Representative Strains for Profiling [Seite 614]
7.11.4 - 21C.4 Selection of Strains against a Background of Their Variable Number Tandem Repeat (VNTR) Designation [Seite 615]
7.11.5 - 21C.5 Potential to Type P. aeruginosa using MALDI-TOF MS [Seite 615]
7.11.6 - 21C.6 Data Processing: Analyzing Data using BioNumerics 7 [Seite 616]
7.11.7 - 21C.7 Discussion and Data Interpretation [Seite 617]
7.11.8 - 21C.8 Going Forward - Reproducibility the Salient Determinant [Seite 621]
7.11.9 - References [Seite 622]
8 - Index [Seite 627]
9 - EULA [Seite 649]
