Mass Spectrometry for Microbial Proteomics

✍ Scribed by Haroun N. Shah, Saheer E. Gharbia

• Publisher: Wiley
• Year: 2010
• Tongue: English
• Leaves: 535
• Edition: 1
• Category: Library

✦ Synopsis

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.

✦ Table of Contents

Mass Spectrometry for Microbial Proteomics......Page 5
Contents......Page 9
Preface......Page 21
List of Contributors......Page 23
Part I: Microbial Characterisation; the Transition from Conventional Methods to Proteomics......Page 27
1.1 Background and Early Attempts to Use Mass Spectrometry on Microbes......Page 29
1.2 Characterisation of Microorganisms by MALDI-TOF-MS; from Initial Ideas to the Development of the First Comprehensive Database......Page 32
1.3.1 A Protein Fingerprinting Platform to Replace SDS-PAGE......Page 35
1.3.2 A Species-Specific Diagnostic Method......Page 37
1.3.3 A Biomarker Search Tool......Page 39
1.4.1 2D GE......Page 44
1.4.2 The DIGE Technique......Page 47
1.5 Nanoparticles as an Alternative Approach in the Analysis and Detection of Low Abundance and Low Molecular Weight Proteins Using MALDI-TOF-MS......Page 51
References......Page 55
2.2 Bacterial Phylogeny: Overview and Key Unresolved Issues......Page 61
2.3 New Protein-Based Molecular Markers for Systematic and Evolutionary Studies......Page 63
2.4 Molecular Markers Elucidating the Evolutionary Relationships among α-Proteobacteria......Page 67
2.5 Molecular Markers for the Bacteroidetes-Chlorobi Phyla......Page 71
2.6 Branching Order and Interrelationships among Bacterial Phyla......Page 72
2.7 Importance of Protein Markers for Discovering Unique Properties for Different Groups of Bacteria......Page 74
2.8 Concluding Remarks......Page 75
References......Page 76
Part II: Proteomics Tools and Biomarker Discovery......Page 81
3.1 Introduction......Page 83
3.2.1 Peptide Mass Fingerprint (PMF)......Page 84
3.2.2 Peptide Fragment Fingerprint......Page 86
3.2.4 False Discovery Rate (FDR)......Page 88
3.2.5 Validating Protein Identifications......Page 89
3.2.6 Reference Database......Page 90
3.3.1 Biomarker Discovery......Page 91
3.3.2 Integrating Genomics with Proteomics......Page 93
References......Page 94
4.1 Introduction......Page 99
4.2.1 MALDI Versus ESI......Page 100
4.2.2 Tandem Mass Spectrometry and Hybrid Mass Spectrometers......Page 101
4.2.3 Fragmentation in Tandem Mass Spectrometry......Page 102
4.3.1 Bottom-Up Proteomics......Page 105
4.3.2 Top-Down Proteomics......Page 106
4.4 Multidimensional Protein Identification......Page 107
4.5 Mass Spectrometry-Based Targeted Protein Quantification and Biomarker Discovery......Page 108
4.5.1 Selected Reaction Monitoring......Page 110
References......Page 112
5.1 Introduction......Page 117
5.2.1 Instrumentation for MALDI-MSI......Page 119
5.3.1 Pharmaceuticals......Page 120
5.3.2 MALDI-MSI and Medicine......Page 121
5.3.3 Biotechnology......Page 127
5.4.1 Microbial MALDI-TOF-MSI......Page 130
5.4.2 Microbial Proteomic Characterisation and Classification via MALDI-TOF-MS and MS/MS......Page 133
5.5 Conclusions......Page 138
References......Page 139
Part III: Protein Samples: Preparation Techniques......Page 143
6.1 Introduction......Page 145
6.2.1 Mechanical Lysis......Page 147
6.2.3 Enzymatic Lysis......Page 149
6.3.1 Removal of Interfering Substances......Page 150
6.3.2 Solubilisation Strategies......Page 152
6.3.3 Sample Preparation for Difference in Gel Electrophoresis (DIGE)......Page 153
6.3.4 Preparation of Environmental Samples......Page 155
6.4.2 Secreted Proteins......Page 156
6.5.1 Brief Background to Protein Identification by LC-MS......Page 157
6.5.3 General Sample Lysis Consideration......Page 158
6.5.4 Crude Protein Purification......Page 159
6.5.5 Protein Resolubilsation and In-Solution Digestion......Page 160
6.5.6 Protein Digestion......Page 161
6.5.7 Microscale Clean Up Prior to LC-MS......Page 163
6.6 Conclusion......Page 164
References......Page 165
7.1 Introduction......Page 169
7.1.3 The Spore Structure......Page 170
7.1.4 C. difficile and Disease......Page 171
7.2.1 Sporulation Media......Page 172
7.2.3 Spore Protein Extraction and Solubilisation......Page 173
References......Page 180
8.1 Introduction......Page 183
8.2 The Surface Proteome......Page 184
8.3 Proteomics of Pathogenic Bacteria......Page 185
8.4 Lipid-Based Protein Immobilization Technology......Page 188
8.5 Salmonella Typhimurium – Disease Mechanism and Outer Membrane Proteins......Page 192
8.6 Outer Membrane Proteins of S. Typhimurium......Page 193
8.7 Helicobacter pylori – Disease Mechanism and Outer Membrane Proteins......Page 194
8.8 Surface Proteins of Intact H. pylori......Page 196
References......Page 197
9.1 Introduction......Page 201
9.2 Fractionation as a Means to Decipher Proteome Complexity......Page 202
9.2.1 Subcellular Fractionation......Page 203
9.2.3 Immunoprecipitation......Page 204
9.2.4 Chromatographic Fractionation......Page 205
9.2.5 Electrokinetic Methods in Proteome Fractionation......Page 206
9.3.1 Depletion of a Few High Abundance Proteins......Page 208
9.3.3 Reduction of the Dynamic Concentration Range with Combinatorial Ligand Libraries......Page 210
References......Page 224
10.1.1 State of the Art in Protein Analysis......Page 231
10.1.3 Concept of 3D-Gel Electrophoresis......Page 232
10.2.2 Thermal Management of the 3D-Gel......Page 234
10.2.3 Online Detection of Laser-Induced Fluorescence......Page 236
10.2.5 Sample Preparation and Fluorescent Labelling......Page 237
10.2.6 Sample Loading......Page 238
10.2.7 Image Processing and Data Evaluation......Page 240
10.3.1 Comparison of 3D-Gel with Standard Slab Gel Separation......Page 242
10.3.2 Applications of 3D-Gel Electrophoresis......Page 243
Acknowledgements......Page 245
References......Page 246
Part IV: Characterisation of Microorganisms by Pattern Matching of Mass Spectral Profiles and Biomarker Approaches Requiring Minimal Sample Preparation......Page 249
11.1 Introduction......Page 251
11.2.1 Hepatitis B and C......Page 265
11.2.2 Severe Acute Respiratory Syndrome (SARS)......Page 266
11.2.4 Human T-Cell Leukaemia Virus Type-1 (HTLV-1)......Page 267
11.2.6 Cytomegalovirus (CMV)......Page 268
11.3.2 Fasciolosis......Page 269
11.4.2 Infectious Endocarditis......Page 270
11.4.3 Respiratory Diseases......Page 271
11.4.4 Intra-Amniotic Infection......Page 272
11.4.5 Bacterial Peritonitis......Page 274
11.6 Conclusions......Page 275
References......Page 276
12.1 Identification of Microorganisms in Clinical Routine......Page 281
12.2 Mass Spectrometry and Microbiology......Page 282
12.3 Mass Spectral ‘Fingerprints’ of Whole Cells......Page 283
12.4 Reproducibility of Mass Spectral Fingerprints......Page 286
12.5 Species and Strain Discrimination by Mass Spectrometry......Page 287
12.6 Pattern Matching Approaches for Automated Identification......Page 290
12.7 Mass Spectral Identification of Microorganisms – Requirements for Routine Diagnostics......Page 291
12.8 Automated Mass Spectral Analysis of Microorganisms in Clinical Routine Diagnostics......Page 293
Acknowledgements......Page 295
References......Page 296
Part V: Targeted Molecules and Analysis of Specific Microorganisms......Page 303
13.1 Introduction......Page 305
13.3 Reproducibility......Page 312
13.3.1 Factors Concerning the Sample......Page 313
13.3.2 Factors Concerning the MALDI MS Process......Page 317
13.4.1 Bacillus spp.......Page 326
13.4.2 Staphylococcus spp.......Page 336
13.4.3 Streptococcus spp.......Page 337
13.4.4 Mycobacterium spp.......Page 338
13.4.5 Other Gram-Positive Bacteria......Page 339
13.4.6 Escherichia coli......Page 341
13.4.7 Gram-Negative Food- and Waterborne Pathogen Proteobacteria Other Than E. coli......Page 342
13.4.8 Typical Sexually Transmitted Pathogens: Neisseria spp. and Haemophilus spp.......Page 344
13.4.9 Gram-Negative Biothreat Agent Bacteria......Page 345
13.4.10 Other Gram-Negative Bacteria......Page 346
13.4.11 Pathogenic Cyanobacteria......Page 348
13.5.1 Protein Database Consideration......Page 349
13.5.2 On-Target Treatment and Analysis......Page 350
13.5.3 ‘Off-Target’ Analysis and Correlation with Proteomics Studies......Page 351
13.6 Conclusions and Outlook......Page 352
References......Page 353
14.1 Introduction......Page 365
14.2.1 Protein Export Machineries of B. subtilis......Page 366
14.2.2 The Extracellular Proteome of B. subtilis......Page 367
14.2.3 The Cell Wall Proteome of B. subtilis......Page 368
14.2.4 The Membrane Attached Lipoproteome of B. subtilis......Page 369
14.2.5 The Proteome Analysis of Protein Secretion Mechanisms in B. subtilis......Page 370
14.3.1 Proteomic Signatures of B. subtilis in Response to Stress and Starvation......Page 375
14.3.2 Proteomic Signatures of B. subtilis in Response to Thiol-Reactive Electrophiles Uncovered the Novel MarR-Type Regulators MhqR and Yod B......Page 377
14.3.3 The MarR/DUF 24-Family YodB Repressor is Directly Sensing Thiol-Reactive Electrophiles via the Conserved Cys6 Residue......Page 379
14.4.1 The Thiol-Redox Proteome of B. subtilis in Response to Diamide and Quinones......Page 380
14.4.2 Depletion of Thiol-Containing Proteins by Quinones Due toThiol-(S)-Alkylation......Page 382
14.5 Proteomics as a Tool to Define Regulons and Targets for Noncoding RNAs......Page 383
References......Page 387
15.1 Introduction to Molecular Pathogenesis of Francisella tularensis Infection......Page 393
15.2.1 Introduction......Page 395
15.2.2 Experimental Procedure......Page 396
15.2.3 Results and Discussion......Page 398
15.3.1 Introduction......Page 401
15.3.2 Analysis of Oxidative Phosphorylation Membrane Protein Complexes of F. tularensis by 2D BN/SDS-PAGE......Page 403
15.4.1 Introduction......Page 408
15.4.2 Methods......Page 409
15.4.3 Results......Page 411
15.4.4 Discussion......Page 412
15.5.1 Introduction......Page 413
15.5.3 Results and Discussion......Page 414
15.5.4 Conclusion......Page 415
References......Page 416
16.1 Introduction......Page 421
16.2 Comparative Genomics......Page 423
16.3 Transcriptomics......Page 424
16.4 Proteomics and Immmunoproteomics......Page 425
16.6 Meningococcal Vaccines and Reverse Vaccinology......Page 427
16.7 Helicobacter pylori Vaccines......Page 428
16.8 Conclusions......Page 429
References......Page 430
Part VI: Statistical Analysis of 2D Gels and Analysis of Mass Spectral Data......Page 435
17.1 Introduction......Page 437
17.2.1 Noise Reduction, Baseline Removal and Normalization......Page 438
17.2.2 Feature Selection......Page 439
17.3 Classification of MS Data......Page 440
17.3.1 Clustering......Page 441
17.3.2 Support Vector Machines......Page 442
17.3.3 Decision Trees......Page 443
17.3.5 Artificial Neural Networks......Page 444
17.4 Evaluation of Classification Models......Page 445
References......Page 446
18.1 Introduction......Page 449
18.2.1 How Many Measurements/Observations are Needed?......Page 450
18.2.2 What Type of Repeat Measurement Should be Used?......Page 452
18.2.3 Sampling Depth......Page 454
18.3.1 Understanding the Statistical Tests: Is It Statistically Significant?......Page 455
18.3.2 Understanding the Statistical Tests: The Hypothesis Test Outcomes......Page 457
18.3.4 Understanding the Statistical Tests: The Multiple Testing Problem......Page 458
18.3.5 Understanding the Statistical Tests: The Assumptions......Page 460
18.4 Validation......Page 462
18.5 Conclusions......Page 463
References......Page 464
Part VII: DNA Resequencing by MALDI-TOF-Mass Spectrometry and Its Application to Traditional Microbiological Problems......Page 467
19.1 Introduction......Page 469
19.2 Comparative Sequence Analysis by MALDI-TOF MS......Page 473
19.3 Applications of Nucleic Acid Analysis by MALDI-TOF MS in Clinical Microbiology......Page 481
19.4 Conclusion......Page 485
References......Page 486
20.1 Introduction......Page 489
20.2.1 Biology......Page 490
20.2.3 Clinical Disease......Page 491
20.3.2 Virulence and Gene Transfer......Page 492
20.3.3 Necessity to Subtype......Page 493
20.4.1 Serotyping......Page 494
20.4.3 Flagellar Variation......Page 495
20.4.4 Somatic Antigens......Page 497
20.5 Sequence-Based Methods to Determine Serotypes......Page 501
20.5.2 Specific SNPs......Page 503
20.5.4 Variation of the Rfb Genes......Page 504
20.6 Transferring to a MALDI Platform for Rapid Analysis......Page 505
20.6.1 Different Methods Available......Page 506
20.6.4 Gene Selection......Page 507
20.6.6 Clustering and Sequence Variation of Amplicons......Page 508
20.7 Conclusions and Summary......Page 514
References......Page 516
Index......Page 523