Haroun N. Shah led the establishment of unique laboratory capabilities, transforming Public Health Laboratory Services' identification of new and emerging threats through mass spectrometry combined with molecular technologies between 1999-2015. After his retirement, he continued to provide expert advice and training to industry and academia to advance innovations and embed new applications of proteomics across biosciences.

Saheer E. Gharbia is the Deputy Director of Gastrointestinal Infection and Food Safety for the UK Health Security Agency and has led the COVID-19 Genomics Programme to support the response to the COVID-19 pandemic. She continues to develop tools for the analysis and interpretation of complex biological and pathogenic traits and works across the One Health Scientific Community to embed common surveillance mechanisms to detect and track emerging threats.

Ajit J. Shah is a Professor in Bioanalytical Science in the Department of Natural Science, Middlesex University, UK.

Erika Y. Tranfield is Scientific Affairs Manager Microbiology at Bruker.

K. Clive Thompson is Chief Scientist at ALS, Life Sciences, UK, an analytical testing organisation in the UK and Ireland.

List of Contributors xix Preface xxiii 1 Progress in the Microbiological Applications of Mass Spectrometry: from Electron Impact to Soft Ionization Techniques, MALDI- TOF MS and Beyond 1Emmanuel Raptakis, Ajit J. Shah, Saheer E. Gharbia, Laila M.N. Shah, Simona Francese, Erika Y. Tranfield, Louise Duncan, and Haroun N. Shah 1.1 Introduction 1 1.1.1 Algorithms Based upon Traditional Carbohydrate Fermentation Tests 1 1.1.2 Dynamic Changes in the Chemotaxonomic Era (c. 1970-1985) through the Lens of the Genus Bacteroides 2 1.1.3 Microbial Lipids as Diagnostic Biomarkers; Resurgence of Interest in MALDI- TOF MS with Advances in Lipidomics 3 1.2 The Dawn of MALDI- TOF MS: Establishing Proof of Concept for Diagnostic Microbiology 7 1.2.1 Development of a MALDI- TOF MS Database for Human Infectious Diseases 10 1.2.2 The Dilemma with Clostridium difficile: from Intact Cells to Intracellular Proteins, MALDI- TOF MS Enters a New Phase 13 1.3 Linear/Reflectron MALDI- TOF MS to Tandem Mass Spectrometry 15 1.3.1 Tandem MALDI- TOF Mass Spectrometry 17 1.3.2 Electrospray- based Mass Analysers 18 1.3.3 Tandem Mass Spectrometry 18 1.3.4 Mass Spectrometry- based Proteomics 19 1.3.5 Case Study: LC- MS/MS of Biothreat Agents, Proteomes of Pathogens and Strain- level Tying Using Bottom- up and Top- down Proteomics 19 1.3.6 Discovery Proteomics 21 1.3.7 Targeted Proteomics 22 1.3.8 Top- down Proteomics 23 1.3.9 Targeted Protein Quantitation 24 1.4 The Application of MALDI- MS Profiling and Imaging in Microbial Forensics: Perspectives 25 1.4.1 MALDI- MSP of Microorganisms and their Products 26 1.5 Hydrogen/Deuterium Exchange Mass Spectrometry in Microbiology 27 1.6 The Omnitrap, a Novel MS Instrument that Combines Many Applications of Mass Spectrometry 29 References 35 2 Machine Learning in Analysis of Complex Flora Using Mass Spectrometry 45Luis Mancera, Manuel J. Arroyo, Gema Méndez, Omar Belgacem, Belén Rodríguez-Sánchez, and Marina Oviaño 2.1 Introduction 45 2.2 An Improved MALDI- TOF MS Data Analysis Pipeline for the Identification of Carbapenemase- producing Klebsiella pneumoniae 47 2.2.1 Motivation 47 2.2.2 Materials and Methods 47 2.2.3 Spectra Acquisition 50 2.2.4 Results 51 2.2.5 Discussion 54 2.3 Detection of Vancomycin- Resistant Enterococcus faecium 55 2.3.1 Motivation 55 2.3.2 Materials and Methods 56 2.3.3 Results and Discussion 59 2.4 Detection of Azole Resistance in Aspergillus fumigatus Complex Isolates 59 2.4.1 Introduction 59 2.4.2 Material and Methods 60 2.4.3 Results 60 2.4.4 Discussion 64 2.5 Peak Analysis for Discrimination of Cryptococcus neoformans Species Complex and their Interspecies Hybrids 64 2.5.1 Motivation 64 2.5.2 Material and Methods 65 2.5.3 Results and Discussion 65 2.6 Conclusions 66 References 67 3 Top- down Identification of Shiga Toxin (and Other Virulence Factors and Biomarkers) from Pathogenic E. coli using MALDI- TOF/TOF Tandem Mass Spectrometry 71Clifton K. Fagerquist 3.1 Introduction 71 3.2 Decay of Metastable Peptide and Protein Ions by the Aspartic Acid Effect 72 3.3 Energy Deposition during Desorption/Ionization by MALDI 75 3.4 Protein Denaturation and Fragmentation Efficiency of PSD 76 3.5 Arginine and its Effect on Fragment Ion Detection and MS/MS Spectral Complexity 79 3.6 Inducing Gene Expression in Wild- type Bacteria for Identification by Top- Down Proteomic Analysis 82 3.7 Top- down Proteomic Identification of B- Subunit of Shiga Toxin from STEC Strains 83 3.8 Furin- digested Shiga Toxin and Middle- down Proteomics 85 3.9 Top- down Identification of an Immunity Cognate of a Bactericidal Protein Produced from a STEC Strain 87 3.10 Lc- Maldi- Tof/tof 88 3.11 Conclusions 89 References 94 4 Liquid Atmospheric Pressure (LAP) - MALDI MS(/MS) Biomolecular Profiling for Large- scale Detection of Animal Disease and Food Adulteration and Bacterial Identification 97Cristian Piras and Rainer Cramer 4.1 Introduction 97 4.2 Background to LAP- MALDI MS 98 4.3 Bacterial Identification by LAP- MALDI MS 102 4.4 Food Adulteration and Milk Quality Analysis by LAP- MALDI MS 105 4.5 Animal Disease Detection by LAP- MALDI MS 108 4.6 Antibiotic Resistance Detection of Microbial Consortia by Lap- Maldi Ms 110 4.7 Future Directions for LAP- MALDI MS Applications 113 References 114 5 Development of a MALDI- TOF Mass Spectrometry Test for Viruses 117Ray K. Iles, Jason K. Iles, and Raminta Zmuidinaite 5.1 Introduction 117 5.2 Understanding the Systems Biology of the Virus and Viral Infections 120 5.3 Understanding the Nature of Viral Proteins and Molecular Biology 121 5.4 Virion Protein Solubilization and Extraction 123 5.5 Sampling and Virion Enrichment 123 5.6 Peak Identification: Quantification and Bioinformatics 125 5.7 Promise and Pitfalls of Machine Learning Bioinformatics 126 5.8 Accelerating MALDI- TOF Assay Protocol Development Using Pseudotypes/ pseudoviruses 128 5.9 Understanding the Operational Parameters of your MALDI- TOF MS 130 5.10 Understanding the Operational Requirements of the Clinical Testing Laboratory: Validation and International Accreditation 131 5.10.1 Limitation and Advantages of CLIA LDTs 131 5.11 MALDI- TOF MS Screening Test for SARS- CoV- 2s 132 5.11.1 Prepare Positive Control 132 5.11.2 Prepare Gargle- saliva Samples 132 5.11.3 Viral Particle Enrichment 132 5.11.4 Dissolution of Virions and Solubilization of Viral Proteins 133 5.11.5 Maldi- Tof Ms 133 5.12 CLIA LDT Validation of a MALDI- TOF MS Test for SARS- CoV- 2 133 5.12.1 Limit of Detection 134 5.12.2 Interfering Substances and Specificity 134 5.12.3 Clinical Performance Evaluation 136 5.12.3.1 Establishing Operational Cut- off Values 137 5.12.3.2 Direct comparison with an RT- PCR SARS- CoV- 2 test 138 5.12.3.3 Internal Sampling Quality Control 138 5.12.3.4 Daily System Quality Control 138 5.12.4 Reproducibility 139 5.12.5 Stability 139 5.12.6 Validation Disposition 141 5.12.6.1 Global Biosecurity 141 References 142 6 A MALDI- TOF MS Proteotyping Approach for Environmental, Agricultural and Food Microbiology 147Hiroto Tamura 6.1 Introduction 147 6.2 Serotyping of Salmonella enterica Subspecies enterica 151 6.3 Discrimination of the Lineages of Listeria monocytogenes and Species of Listeria 161 6.4 Discrimination of the Bacillus cereus Group and Identification of Cereulide 167 6.5 Identification of Alkylphenol Polyethoxylate- degrading Bacteria in the Environment 171 6.6 Conclusions and Future Perspectives 173 References 175 7 Diversity, Transmission and Selective Pressure on the Proteome of Pseudomonas aeruginosa 183Louise Duncan, Ajit J. Shah, Malcolm Ward, Radhey S. Gupta, Bashudev Rudra, Alvin Han, Ken Bruce, and Haroun N. Shah 7.1 Introduction: Diversity 183 7.1.1 P. aeruginosa: from 'Atypical' to Diverse 183 7.1.2 Phenotypical Diversity in Isolates from Different Environments 183 7.1.2.1 Clinical Isolates 183 7.1.2.2 Environmental Isolates 184 7.1.2.3 Veterinary Isolates 184 7.1.2.4 Comparing P. aeruginosa Phenotypical Profiles from Different Environments 184 7.1.2.5 Antibiotic Resistance in P. aeruginosa from Different Environments 186 7.1.3 The Relationship Between Phenotypical and Proteomic Diversity 186 7.1.4 Techniques and Practical Considerations for Studying Proteomic Diversity 186 7.1.5 Proteomic Diversity and MS Applications 189 7.2 Transmission 189 7.2.1 The History of P. aeruginosa Transmission 189 7.2.2 Proteomics and P. aeruginosa Transmission 191 7.2.3 The Impact of Proteomic Diversity on Transmission 191 7.3 Selective Pressures on the Proteome 192 7.3.1 Tandem MS Systems for Studying Selected Proteomes 192 7.3.2 Microenvironment Selection 192 7.3.2.1 The Human Body and CF Lung 192 7.3.2.2 The Natural Environment 192 7.3.3 Antimicrobial Selection 193 7.4 Conclusions on Studies of the Proteome 193 7.5 Genomic Studies on Pseudomonas aeruginosa Strains Revealing the Presence of Two Distinct Clades 195 7.5.1 Phylogenomic Analysis Reveals the Presence of Two Distinct Clades Within P. aeruginosa 196 7.5.2 Identification of Molecular Markers Distinguishing the Two P. aeruginosa Clades 198 7.6 Final Conclusions 201 References 201 8 Characterization of Biodegradable Polymers by MALDI- TOF MS 211Hiroaki Sato 8.1 Introduction 211 8.2 Structural Characterization of Poly(¿- caprolactone) Using Maldi- Tof Ms 212 8.3 Biodegradation Profiles of a Terminal- modified PCL Observed by Maldi- Tof Ms 216 8.4 Bacterial Biodegradation Mechanisms of Non- ionic Surfactants 218 8.5 Advanced Molecular Characterization by High- resolution MALDI- TOF MS Combined with KMD Analysis 221 8.6 Structural Characterization of High- molecular- weight Biocopolyesters by High- resolution MALDI- TOF MS Combined with KMD Analysis 225 References 228 9 Phytoconstituents and Antimicrobiological Activity 231Philip L. Poole and Giulia T.M. Getti 9.1 Introduction to Phytochemicals 231 9.2 An Application to Bacteriology 233 9.2.1 Allicin Leads to a Breakdown of the Cell Wall of Staphylococcus aureus 234 9.3 Applications to Parasitology 239 9.3.1 Drug Discovery 239 9.3.2 Parasite Characterization 240 9.4 A Proteomic Approach: Leishmania Invasion of Macrophages 240 9.5 Intracellular Leishmania Amastigote Spreading between Macrophages 243 9.6 Potential Virus Applications 244 Acknowledgements 246 References 246 10 Application of MALDI- TOF MS in Bioremediation and Environmental Research 255Cristina Russo and Diane Purchase 10.1 Introduction 255 10.2 Microbial Identification: Molecular Methods and MALDI- TOF MS 257 10.2.1 PCR- based Methods 258 10.2.2 Maldi- Tof Ms 260 10.3 Combination of MALDI- TOF MS with Other Methods for the Identification of Microorganisms 261 10.4 Application of MALDI- TOF MS in Environmental and Bioremediation Studies 263 10.4.1 The Atmospheric Environment 263 10.4.2 The Aquatic Environment 263 10.4.3 The Terrestrial Environment 265 10.4.4 Bioremediation Research Applications 266 10.5 Microbial Products and Metabolite Activity 268 10.6 Challenges of Environmental Applications 270 10.7 Opportunities and Future Outlook 271 10.8...