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Book SynopsisMicroproteins: Uncovering Hidden Layers of the Proteome.- Proteomic Detection of Small and Alternative Open Reading Frame-Encoded Microproteins and Alternative Proteins.- Identification of Canonical and Alternative Proteins by Mass Spectrometry.- Identification and Characterization of Microproteins/MicroORFs.- Proteogenomic Identification of Annotated and Novel smORFs via Microprotein Enrichment.- Identification of Peptides Encoded by Small Open Reading Frames in Microalgae by Mass Spectrometry.- Identifying and Visualizing Novel Small Open Reading Frames (sORFs) via Ribo-Seq, Transcriptome Assembly, and ggRibo.- Translation Signature Scores: Data-Driven Approach to Assess Evidence for Active Translation.- Identification of Potentially Coding Small ORFs in Plant Transcriptomes.- Structure and Disorder Predictions of Microproteins: Usage, Applications, and Pitfalls.- Computational Methods for Tracing the Evolutionary History of Human Microproteins Encoded by Intronless Genes.- Bioinformatic Tools for Non-Bioinformaticians: A Roadmap to Study Unknown Microprotein Sequences.- Functional Testing of Microproteins in a Vertebrate Model of Development.- Functional Validation of Transcriptionally Active Microproteins/sORFs In Planta.- Elucidating the Localization and Interactome of Mitochondrial Microproteins.- Illuminating Small Proteins through HiBiT Blotting.- Gain-of-Functional Genomic Screening for Microproteins Essential for Tumorigenesis.- Proteomic Identification of Microproteins Translated from Long Non-Coding RNA.- Urinary Micropeptides: Integrative In Silico Profiling and LC-MS/MS Analysis for Prostate Cancer Biomarker Discovery.- Using Untargeted Mass Spectrometry-Based Proteomics to Identify Putative Microprotein Biomarkers from Patient Tampons.

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Book SynopsisAddressing key aspects of cancer research from an interdisciplinary perspective and presenting analytical, quantitative and computational models, this book is a pedagogical introduction to cancer for physics students and researchers, as well as a reference text for cancer biologists interested in quantitative tools and approaches from physics.Trade Review'The Physics of Cancer explores a growing field and seeks to incorporate discussions pertaining to both the physics and biology of cancer. [The authors] provide readers with a refreshing perspective on cancer research. The volume not only offers a clear description of the fundamental biological concepts of cancer development and treatment, but also introduces readers with a physics background to the developing interdisciplinary field of the physics of cancer. This new field contributes to the understanding of many aspects of cancer studies, which include cancer stem cell phenotypic switching, cell sorting, mechanics of cancer cells, cell migration, cancer metastasis, etc. Due to the advanced nature of the topic, this book will serve as a good introductory level resource for physics researchers and graduate students who are interested in learning about this interdisciplinary field.' H. Zhou, Choice'La Porta and Zapperi's book isn't just light reading for curious physicists - it can also serve to guide interested researchers into a rich interdisciplinary area.' Guido D'Amico, CERN CourierTable of ContentsPreface; 1. Introduction to the cell; 2. The biology of cancer; 3. A modeling toolbox for cancer growth; 4. Vascular hydrodynamics and tumor angiogenesis; 5. Cancer stem cells and the population dynamics of tumors; 6. Biomechanics of cancer; 7. Cancer cell migration; 8. Chromosome and chromatin dynamics in cancer; 9. Control of tumor growth by the immune system; 10. Pharmacological approaches: old and new; 11. Outlook on the physics of cancer: a new interdisciplinary area; References; Index.

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Table of ContentsList of Contributors xiii Foreword xvii Preface xix 1 Contemporary Protein Analysis by Ion Mobility Mass Spectrometry 1Johannes P.C. Vissers and James I. Langridge 1.1 Introduction 1 1.2 Traveling-Wave Ion Mobility Mass Spectrometry 1 1.3 IM–MS and LC–IM–MS Analysis of Simple and Complex Mixtures 2 1.4 Outlook 7 Acknowledgment 8 References 8 2 High-Resolution Accurate Mass Orbitrap and Its Application in Protein Therapeutics Bioanalysis 11Hongxia Wang and Patrick Bennett 2.1 Introduction 11 2.2 Triple Quadrupole Mass Spectrometer and Its Challenges 11 2.3 High-Resolution Mass Spectrometers 12 2.4 Quantitation Modes on Q Exactive Hybrid Quadrupole Orbitrap 13 2.5 Protein Quantitation Approaches Using Q Exactive Hybrid Quadrupole Orbitrap 14 2.6 Data Processing 16 2.7 Other Factors That Impact LC–MS-based Quantitation 16 2.8 Conclusion and Perspectives of LC–HRMS in Regulated Bioanalysis 18 References 18 3 Current Methods for the Characterization of Posttranslational Modifications in Therapeutic Proteins Using Orbitrap Mass Spectrometry 21Zhiqi Hao, Qiuting Hong, Fan Zhang, Shiaw-Lin Wu, and Patrick Bennett 3.1 Introduction 21 3.2 Characterization of PTMs Using Higher-Energy Collision Dissociation 23 3.3 Application of Electron Transfer Dissociation to the Characterization of Labile PTMs 26 3.4 Conclusion 31 Acknowledgment 32 References 32 4 Macro- to Micromolecular Quantitation of Proteins and Peptides by Mass Spectrometry 35Suma Ramagiri, Brigitte Simons, and Laura Baker 4.1 Introduction 35 4.2 Key Challenges of Peptide Bioanalysis 36 4.3 Key Features of LC/MS/MS-Based Peptide Quantitation 38 4.4 Advantages of the Diversity of Mass Spectrometry Systems 41 4.5 Perspectives for the Future 41 References 42 5 Peptide and Protein Bioanalysis Using Integrated Column-to-Source Technology for High-Flow Nanospray 45Shane R. Needham and Gary A. Valaskovic 5.1 Introduction – LC–MS Has Enabled the Field of Protein Biomarker Discovery 45 5.2 Integration of Miniaturized LC with Nanospray ESI-MS Is a Key for Success 46 5.3 Micro- and Nano-LC Are Well Suited for Quantitative Bioanalysis 47 5.4 Demonstrating Packed-Emitter Columns Are Suitable for Bioanalysis 49 5.5 Future Outlook 51 References 52 6 Targeting the Right Protein Isoform: Mass Spectrometry-Based Proteomic Characterization of Alternative Splice Variants 55Jiang Wu 6.1 Introduction 55 6.2 Alternative Splicing and Human Diseases 55 6.3 Identification of Splice Variant Proteins 56 6.4 Conclusion 64 References 64 7 The Application of Immunoaffinity-Based Mass Spectrometry to Characterize Protein Biomarkers and Biotherapeutics 67Bradley L. Ackermann and Michael J. Berna 7.1 Introduction 67 7.2 Overview of IA-MS Methods 69 7.3 IA-MS Applications – Biomarkers 74 7.3.1 Peptide Biomarkers 74 7.4 IA-MS Applications – Biotherapeutics 81 7.5 Future Direction 84 References 85 8 Semiquantification and Isotyping of Antidrug Antibodies by Immunocapture-LC/MS for Immunogenicity Assessment 91Jianing Zeng, Hao Jiang, and Linlin Luo 8.1 Introduction 91 8.2 Multiplexing Direct Measurement of ADAs by Immunocapture-LC/MS for Immunogenicity Screening, Titering, and Isotyping 93 8.3 Indirect Measurement of ADAs by Quantifying ADA Binding Components 95 8.4 Use of LC–MS to Assist in Method Development of Cell-Based Neutralizing Antibody Assays 96 8.5 Conclusion and Future Perspectives 97 References 97 9 Mass Spectrometry-Based Assay for High-Throughput and High-Sensitivity Biomarker Verification 99Xuejiang Guo and Keqi Tang 9.1 Background 99 9.2 Sample Processing Strategies 100 9.3 Advanced Electrospray Ionization Mass Spectrometry Instrumentation 102 9.4 Conclusion 105 References 105 10 Monitoring Quality of Critical Reagents Used in Ligand Binding Assays with Liquid Chromatography Mass Spectrometry (LC–MS) 107Brian Geist, Adrienne Clements-Egan, and Tong-Yuan Yang 10.1 Introduction 107 10.2 Case Study Examples 114 10.3 Discussion 122 Acknowledgment 126 References 126 11 Application of Liquid Chromatography-High Resolution Mass Spectrometry in the Quantification of Intact Proteins in Biological Fluids 129Stanley (Weihua) Zhang, Jonathan Crowther, and Wenying Jian 11.1 Introduction 129 11.2 Workflows for Quantification of Proteins Using Full-Scan LC-HRMS 131 11.3 Internal Standard Strategy 133 11.4 Calibration and Quality Control (QC) Sample Strategy 135 11.5 Common Issues in Quantification of Proteins Using LC-HRMS 135 11.6 Examples of LC-HRMS-Based Intact Protein Quantification 137 11.7 Conclusion and Future Perspectives 138 Acknowledgment 140 References 140 12 LC–MS/MS Bioanalytical Method Development Strategy for Therapeutic Monoclonal Antibodies in Preclinical Studies 145Hongyan Li, Timothy Heath, and Christopher A. James 12.1 Introduction: LC-MS/MS Bioanalysis of Therapeutic Monoclonal Antibodies 145 12.2 Highlights of Recent Method Development Strategies 146 12.3 Case Studies of Preclinical Applications of LC–MS/MS for Monoclonal Antibody Bioanalysis 154 12.4 Conclusion and Future Perspectives 156 References 158 13 Generic Peptide Strategies for LC–MS/MS Bioanalysis of Human Monoclonal Antibody Drugs and Drug Candidates 161Michael T. Furlong 13.1 Introduction 161 13.2 A Universal Peptide LC–MS/MS Assay for Bioanalysis of a Diversity of Human Monoclonal Antibodies and Fc Fusion Proteins in Animal Studies 161 13.3 An Improved “Dual” Universal Peptide LC–MS/MS Assay for Bioanalysis of Human mAb Drug Candidates in Animal Studies 165 13.4 Extending the Universal Peptide Assay Concept to Human mAb Bioanalysis in Human Studies 170 13.5 Internal Standard Options for Generic Peptide LC–MS/MS Assays 173 13.6 Sample Preparation Strategies for Generic Peptide LC–MS/MS Assays 175 13.7 Limitations of Generic Peptide LC–MS/MS Assays 177 13.8 Conclusion 178 Acknowledgments 178 References 178 14 Mass Spectrometry-Based Methodologies for Pharmacokinetic Characterization of Antibody Drug Conjugate Candidates During Drug Development 183Yongjun Xue, Priya Sriraman, Matthew V. Myers, Xiaomin Wang, Jian Chen, Brian Melo, Martha Vallejo, Stephen E. Maxwell, and Sekhar Surapaneni 14.1 Introduction 183 14.2 Mechanism of Action 183 14.3 Mass Spectrometry Measurement for DAR Distribution of Circulating ADCs 186 14.4 Total Antibody Quantitation by Ligand Binding or LC–MS/MS 189 14.5 Total Conjugated Drug Quantitation by Ligand Binding or LC–MS/MS 193 14.6 Catabolite Quantitation by LC–MS/MS 196 14.7 Preclinical and Clinical Pharmacokinetic Support 197 14.8 Conclusion and Future Perspectives 198 References 198 15 Sample Preparation Strategies for LC–MS Bioanalysis of Proteins 203Long Yuan and Qin C. Ji 15.1 Introduction 203 15.2 Sample Preparation Strategies to Improve Assay Sensitivity 205 15.3 Sample Preparation Strategies to Differentiate Free, Total, and ADA-Bound Proteins 213 15.4 Sample Preparation Strategies to Overcome Interference from Antidrug Antibodies or Soluble Target 214 15.5 Protein Digestion Strategies 214 15.6. Conclusion 215 Acknowledgment 216 References 216 16 Characterization of Protein Therapeutics by Mass Spectrometry 221Wei Wu, Hangtian Song, Thomas Slaney, Richard Ludwig, Li Tao, and Tapan Das 16.1 Introduction 221 16.2 Variants Associated with Cysteine/Disulfide Bonds in Protein Therapeutics 221 16.3 N–C-Terminal Variants 225 16.4 Glycation 226 16.5 Oxidation 226 16.6 Discoloration 228 16.7 Sequence Variants 230 16.8 Glycosylation 232 16.9 Conclusion 240 References 240 Index 251

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Book SynopsisIt has long been recognized that metal spin states play a central role in the reactivity of important biomolecules, in industrial catalysis and in spin crossover compounds. As the fields of inorganic chemistry and catalysis move towards the use of cheap, non-toxic first row transition metals, it is essential to understand the important role of spin states in influencing molecular structure, bonding and reactivity. Spin States in Biochemistry and Inorganic Chemistry provides a complete picture on the importance of spin states for reactivity in biochemistry and inorganic chemistry, presenting both theoretical and experimental perspectives. The successes and pitfalls of theoretical methods such as DFT, ligand-field theory and coupled cluster theory are discussed, and these methods are applied in studies throughout the book. Important spectroscopic techniques to determine spin states in transition metal complexes and proteins are explained, and the use of NMR for the analyTrade Review"Spin States in Biochemistry and Inorganic Chemistry: Influence on Structure and Reactivity, edited by Marcel Swart and Miquel Costas is impressive testimony to the advances in theory, computations, and experiment, especially regarding transition metals in recent years, and a revealing look at how much remains to be developed....The authors provide detailed comparison of various computational methods with each other and with experimental data in many cases. Each chapter is an extensively referenced focused review article. Chapters 1-3 emphasize computational methods....No single monograph can encompass a topic as broad as the title of this book, which is almost the entire chemistry of the periodic table. However, for the selected topics, the volume provides a very valuable concise snapshot of the field.Computational chemistry for compounds of CHNO have advanced to the point that many experimentalists can routinely apply standard methods in Gaussian and other such programs with confidence, guided only by the state of the art described in other publications. This book shows that in spite of enormous effort related to transition metal energy states and spin states, even the expert computational chemists need to proceed with caution and compare many functionals"- (Gareth Eaton- December 2016)Table of ContentsAbout the Editors xv List of Contributors xvii Foreword xxi Acknowledgments xxiii 1 General Introduction to Spin States 1Marcel Swart and Miquel Costas 1.1 Introduction 1 1.2 Experimental Chemistry: Reactivity, Synthesis and Spectroscopy 2 1.3 Computational Chemistry: Quantum Chemistry and Basis Sets 4 2 Application of Density Functional and Density Functional Based Ligand Field Theory to Spin States 7Claude Daul, Matija Zlatar, Maja Gruden-Pavlovic and Marcel Swart 2.1 Introduction 7 2.2 What Is the Problem with Theory? 9 2.2.1 Density Functional Theory 9 2.2.2 LF Theory: Bridging the Gap Between Experimental and Computational Coordination Chemistry 11 2.3 Validation and Application Studies 15 2.3.1 Use of OPBE, SSB-D and S12g Density Functionals for Spin-State Splittings 17 2.3.2 Application of LF-DFT 21 2.4 Concluding Remarks 25 3 Ab Initio Wavefunction Approaches to Spin States 35Carmen Sousa and Coen de Graaf 3.1 Introduction and Scope 35 3.2 Wavefunction-Based Methods for Spin States 35 3.2.1 Single Reference Methods 36 3.2.2 Multireference Methods 37 3.2.3 MR Perturbation Theory 39 3.2.4 Variational Approaches 40 3.2.5 Density Matrix Renormalization Group Theory 40 3.3 Spin Crossover 41 3.3.1 Choice of Active Space and Basis Set 41 3.3.2 The HS–LS Energy Difference 43 3.3.3 Light-Induced Excited Spin State Trapping (LIESST) 45 3.3.4 Spin Crossover in Other Metals 47 3.4 Magnetic Coupling 47 3.5 Spin States in Biochemical and Biomimetic Systems 50 3.6 Two-State Reactivity 52 3.7 Concluding Remarks 52 4 Experimental Techniques for Determining Spin States 59Carole Duboc and Marcello Gennari 4.1 Introduction 59 4.2 Magnetic Measurements 61 4.2.1 g-Anisotropy and Zero-Field Splitting (zfs) 64 4.2.2 Unquenched Orbital Moment in the Ground State 64 4.2.3 Exchange Interactions 64 4.2.4 Spin Transitions and Spin Crossover 66 4.3 EPR Spectroscopy 66 4.4 Mössbauer Spectroscopy 70 4.5 X-ray Spectroscopic Techniques 74 4.6 NMR Spectroscopy 77 4.7 Other Techniques 80 4.A Appendix 81 4.A.1 Theoretical Background 81 4.A.2 List of Symbols 82 5 Molecular Discovery in Spin Crossover 85Robert J. Deeth 5.1 Introduction 85 5.2 Theoretical Background 85 5.2.1 Spin Transition Curves 88 5.2.2 Light-Induced Excited Spin State Trapping 89 5.3 Thermal SCO Systems: Fe(II) 90 5.4 SCO in Non-d6 Systems 93 5.5 Computational Methods 95 5.6 Outlook 98 6 Multiple Spin-State Scenarios in Organometallic Reactivity 103Wojciech I. Dzik, Wesley Böhmer and Bas de Bruin 6.1 Introduction 103 6.2 "Spin-Forbidden" Reactions and Two-State Reactivity 104 6.3 Spin-State Changes in Transition Metal Complexes 107 6.3.1 Influence of the Spin State on the Kinetics of Ligand Exchange 108 6.3.2 Stoichiometric Bond Making and Breaking Reactions 109 6.3.3 Spin-State Situations Involving Redox-Active Ligands 115 6.4 Spin-State Changes in Catalysis 119 6.4.1 Catalytic (Cyclo)oligomerizations 119 6.4.2 Phillips Cr(II)/SiO2 Catalyst 121 6.4.3 SNS–CrCl3 Catalyst 123 6.5 Concluding Remarks 125 7 Principles and Prospects of Spin-States Reactivity in Chemistry and Bioinorganic Chemistry 131Dandamudi Usharani, Binju Wang, Dina A. Sharon and Sason Shaik 7.1 Introduction 131 7.2 Spin-States Reactivity 132 7.2.1 Two-State and Multi-State Reactivity 133 7.2.2 Origins of Spin-Selective Reactivity: Exchange-Enhanced Reactivity and Orbital Selection Rules 137 7.2.3 Considerations of Exchange-Enhanced Reactivity versus Orbital-Controlled Reactivity 140 7.2.4 Consideration of Spin-State Selectivity in H-Abstraction: The Power of EER 142 7.2.5 The Origins of Mechanistic Selection – Why Are C–H Hydroxylations Stepwise Processes? 146 7.3 Prospects of Two-State Reactivity and Multi-State Reactivity 148 7.3.1 Probing Spin-State Reactivity 148 7.3.2 Are Spin Inversion Probabilities Useful for Analyzing TSR? 150 7.4 Concluding Remarks 151 8 Multiple Spin-State Scenarios in Gas-Phase Reactions 157Jana Roithová 8.1 Introduction 157 8.2 Experimental Methods for the Investigation of Metal-Ion Reactions 158 8.3 Multiple State Reactivity: Reactions of Metal Cations with Methane 160 8.4 Effect of the Oxidation State: Reactions of Metal Hydride Cations with Methane 163 8.5 Two-State Reactivity: Reactions of Metal Oxide Cations 164 8.6 Effect of Ligands 171 8.7 Effect of Noninnocent Ligands 174 8.8 Concluding Remarks 177 9 Catalytic Function and Mechanism of Heme and Nonheme Iron(IV)–Oxo Complexes in Nature 185Matthew G. Quesne, Abayomi S. Faponle, David P. Goldberg and Sam P. de Visser 9.1 Introduction 185 9.2 Cytochrome P450 Enzymes 186 9.2.1 Importance of Cytochrome P450 Enzymes 187 9.2.2 P450 Activation of Long-Chain Fatty Acids 188 9.2.3 Heme Monooxygenases and Peroxygenases 188 9.2.4 Catalytic Cycle of Cytochrome P450 Enzymes 188 9.3 Nonheme Iron Dioxygenases 190 9.3.1 Cysteine Dioxygenase 191 9.3.2 AlkB Repair Enzymes 192 9.3.3 Nonheme Iron Halogenases 194 9.4 Conclusions 197 9.5 Acknowledgments 197 10 Terminal Metal–Oxo Species with Unusual Spin States 203Sarah A. Cook, David C. Lacy and Andy S. Borovik 10.1 Introduction 203 10.2 Bonding 204 10.2.1 Bonding Considerations: Tetragonal Symmetry 204 10.2.2 Bonding Considerations: Trigonal Symmetry 205 10.2.3 Methods of Characterization 206 10.3 Case Studies 206 10.3.1 Iron–Oxo Chemistry 206 10.3.2 Manganese–Oxo Chemistry 212 10.3.3 Cautionary Tales: Late Transition Metal Oxido Complexes 217 10.3.4 Effects of Redox Inactive Metal Ions 217 10.3.5 Metal–Oxyl Complexes 218 10.4 Reactivity 218 10.4.1 General Concepts: Proton versus Electron Transfer 218 10.4.2 Spin State and Reactivity 220 10.5 Summary 220 11 Multiple Spin Scenarios in Transition-Metal Complexes Involving Redox Non-Innocent Ligands 229Florian Heims and Kallol Ray 11.1 Introduction 229 11.2 Survey of Non-Innocent Ligands 231 11.3 Identification of Non-Innocent Ligands 232 11.3.1 X-ray Crystallography 232 11.3.2 EPR Spectroscopy 234 11.3.3 Mössbauer Spectroscopy 235 11.3.4 XAS Spectroscopy 236 11.4 Selected Examples of Biological and Chemical Systems Involving Non-Innocent Ligands 237 11.4.1 Copper–Radical Interaction 237 11.4.2 Iron–Radical Interaction 246 11.5 Concluding Remarks 252 12 Molecular Magnetism 263Guillem Aromí, Patrick Gamez and Olivier Roubeau 12.1 Introduction 263 12.2 Molecular Magnetism: Motivations, Early Achievements and Foundations 264 12.3 Molecular Nanomagnets (MNM) 265 12.3.1 Single-Molecule Magnets 266 12.3.2 Single-Chain Magnets (SCM) 268 12.3.3 Single-Ion Magnets (SIM) 271 12.4 Switchable Systems 273 12.4.1 Spin Crossover (SCO) 273 12.4.2 Valence Tautomerism (VT) 273 12.4.3 Charge Transfer (CT) 275 12.4.4 Light-Driven Ligand-Induced Spin Change (LD-LISC) 276 12.4.5 Photoswitching (PS) Through Intermetallic CT 277 12.5 Molecular-Based Magnetic Refrigerants 278 12.5.1 The Magneto-Caloric Effect, Its Experimental Determination and Key Parameters 278 12.5.2 Molecular to Extended Framework Coolers Towards Applications 280 12.6 Quantum Manipulation of the Electronic Spin for Quantum Computing 282 12.6.1 Organic Radicals 283 12.6.2 Transition Metal Clusters 284 12.6.3 Lanthanides as Realization of Qubits 285 12.6.4 Engineering of Molecular Quantum Gates with Lanthanide Qubits 285 12.7 Perspectives Toward Applications and Concluding Remarks 287 13 Electronic Structure, Bonding, Spin Coupling, and Energetics of Polynuclear Iron–Sulfur Clusters – A Broken Symmetry Density Functional Theory Perspective 297Kathrin H. Hopmann, Vladimir Pelmenschikov, Wen-Ge Han Du and Louis Noodleman 13.1 Introduction 297 13.2 Iron–Sulfur Coordination: Geometric and Electronic Structure 298 13.3 Spin Polarization Splitting and the Inverted Level Scheme 300 13.4 Spin Coupling and the Broken Symmetry Method 300 13.5 Electron Localization and Delocalization 301 13.6 Polynuclear Systems – Competing Heisenberg Interactions and Spin-Dependent Delocalization 303 13.7 Preamble to Three Major Topics: Iron–Sulfur–Nitrosyls, Adenosine-5'-Phosphosulfate Reductase, and the Proximal Cluster of Membrane-Bound [NiFe]-Hydrogenase 303 13.7.1 Nonheme Iron Nitrosyl Complexes 303 13.7.2 Adenosine-5'-Phosphosulfate Reductase 310 13.7.3 Proximal Cluster of O2-Tolerant Membrane-Bound [NiFe]-Hydrogenase in Three Redox States 315 13.8 Concluding Remarks 318 13.9 Acknowledgments 319 14 Environment Effects on Spin States, Properties, and Dynamics from Multi-level QM/MM Studies 327Alexander Petrenko and Matthias Stein 14.1 Introduction 327 14.1.1 Environmental Effects 328 14.1.2 Hybrid QM/MM Embedding Schemes 329 14.2 The Quantum Spin Hamiltonian – Linking Theory and Experiment 332 14.3 The Solvent as an Environment 335 14.3.1 Fourier Transform Infrared Spectroscopy 336 14.3.2 Nuclear Magnetic Resonance 336 14.3.3 Electron Paramagnetic Resonance 336 14.4 Effect of Different Levels of QM and MM Treatment 338 14.4.1 Convergence and Caveats at the QM Level 338 14.4.2 Accuracy of the MM Part 341 14.4.3 QM versus QM/MM Methods 341 14.5 Illustrative Bioinorganic Examples 343 14.5.1 Cytochrome P450 343 14.5.2 Hydrogenase Enzymes 349 14.5.3 Photosystem II and the Effect of QM Size 354 14.6 From Static Spin-State Properties to Dynamics and Kinetics of Electron Transfer 357 14.7 Final Remarks and Conclusions 359 14.8 Acknowledgments 362 15 High-Spin and Low-Spin States in {FeNO}7, FeIV=O, and FeIII–OOH Complexes and Their Correlations to Reactivity 369Edward I. Solomon, Kyle D. Sutherlin and Martin Srnec 15.1 Introduction 369 15.2 High- and Low-Spin {FeNO}7 Complexes: Correlations to O2 Activation 372 15.2.1 Spectroscopic Definition of the Electronic Structure of High-Spin {FeNO}7 372 15.2.2 Computational Studies of S = 3/2 {FeNO}7 Complexes and Related {FeO2}8 Complexes 375 15.2.3 Extension to IPNS and HPPD: Implications for Reactivity 377 15.2.4 Correlation to {FeNO}7 S = 1/2 385 15.3 Low-Spin (S = 1) and High-Spin (S = 2) FeIV=O Complexes 386 15.3.1 FeIV=O S = 1 Complexes: π* FMO 386 15.3.2 FeIV=O S = 2 Sites: π* and σ* FMOs 390 15.3.3 Contributions of FMOs to Reactivity 392 15.4 Low-Spin (S = 1/2) and High-Spin (S = 5/2) FeIII–OOH Complexes 396 15.4.1 Spin State Dependence of O–O Bond Homolysis 396 15.4.2 FeIII–OOH S = 1/2 Reactivity: ABLM 398 15.4.3 FeIII–OOH Spin State-Dependent Reactivity: FMOs 399 15.5 Concluding Remarks 401 15.6 Acknowledgments 402 16 NMR Analysis of Spin Densities 409Kara L. Bren 16.1 Introduction and Scope 409 16.2 Spin Density Distribution in Transition Metal Complexes 410 16.3 NMR of Paramagnetic Molecules 412 16.3.1 Chemical Shifts 413 16.3.2 Relaxation Rates 414 16.4 Analysis of Spin Densities by NMR 416 16.4.1 Factoring Contributions to Hyperfine Shifts 416 16.4.2 Relaxation Properties and Spin Density 418 16.4.3 DFT Approaches to Analyzing Hyperfine Shifts 419 16.4.4 Natural Bond Orbital Analysis 420 16.4.5 Application and Practicalities 421 16.5 Probing Spin Densities in Paramagnetic Metalloproteins 422 16.5.1 Heme Proteins 422 16.5.2 Iron-Sulfur Proteins 425 16.5.3 Copper Proteins 427 16.6 Conclusions and Outlook 429 17 Summary and Outlook 435Miquel Costas and Marcel Swart 17.1 Summary 435 17.2 Outlook 436 Index 439

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Book SynopsisCytogenetic Laboratory Management Cytogenetic Laboratory Management Chromosomal, FISH and Microarray-Based Best Practices and Procedures Cytogenetic Laboratory Management: Chromosomal, FISH and Microarray-Based Best Practices and Procedures is a practical guide that describes how to develop and implement best practice processes and procedures in the genetic laboratory setting. The text first describes good laboratory practices, including quality management, design control of tests, and FDA guidelines for laboratory-developed tests, and preclinical validation study designs. The second focus of the book is on best practices for staffing and training, including cost of testing, staffing requirements, process improvement using Six Sigma techniques, training and competency guidelines, and complete training programs for cytogenetic and molecular genetic technologists. The third part of the text provides stepwise standard operating procedures for chromosomalTrade Review"From beginning to end, this book provides relevant concepts, procedures and strategies that give the reader a complete overview of good laboratory practices. In addition to providing an excellent guide for setting up a new clinical lab, Zneimer's book should be considered as a useful guide for any laboratory because it provides a wealth of practical information that can be used on a daily basis. All of the spreadsheets, guides, examples and templates in the book are useful bonus features and represent a valuable legacy of the author's extensive experience." (Phenotype June 2017)Table of ContentsDedication xiii Preface xvii Acknowledgments xix About the Companion Website v Section I: Best Practices for Laboratory Operations 1 1 Guidelines for Good Clinical Laboratory Practice 3 1.1 Physical Facilities 4 1.2 Specimen Transport and Management 5 1.3 Personnel Safety 6 1.4 Laboratory Information System (LIS) 7 1.5 Quality Management 7 1.6 Organization and Personnel 10 1.7 Laboratory Equipment 10 1.8 Testing Operating Procedures 11 1.9 Safety Plan 12 1.10 Biosafety Plan 22 1.11 Chemical Hygiene Plan 31 1.12 Health Insurance Portability and Accountability Act (HIPAA) Incident Plan 55 Appendix 1.A: OSHA’s Form 300 60 Appendix 1.B: OSHA’s Form 300A 61 Appendix 1.C: Information on HMIS and NFPA Labeling Systems used in Laboratories 62 Further Reading 66 More Resources 69 HIPAA Reference 70 2 Quality Management 71 2.1 QC Program 74 2.2 Individualized QC Plan (IQCP) 80 2.3 Standards for Test Records and Reporting 81 2.4 Overview of General Culturing Issues 83 2.5 QI Program 95 2.6 Proficiency Testing 101 2.7 Inspection Preparation 110 2.8 Calibration Verification 112 Further Reading 121 3 Design Control of Tests and FDA Guidelines for Laboratory Developed Tests (LDTs) 125 3.1 Design Control of Tests 125 3.2 FDA Guideline Summary for LDTs 139 Further Reading 157 4 Preclinical Validation Studies 159 4.1 Validation Plans and Protocols 159 4.2 Validation Reports 179 4.3 Example Validation Plan and Report—Analysis of FISH Probes for Chromosome 5 Deletion and Monosomy 181 4.4 Example Validation Plan and Report for the FDA‐Approved Vysis ALK FISH Probe 192 Further Reading 206 5 Reagents, Instruments, and Equipment 209 5.1 Reagents 209 5.2 Instruments and Equipment 227 5.3 IQ, OQ, and PQ Procedures 237 5.4 Example Equipment Process Validation Protocol 245 Further Reading 250 Section II: Best Practices for Staffing and Training 253 6 Cost of Testing and Staffing Requirements 255 6.1 Labor Costs 256 6.2 Time and Cost Assessment 260 6.3 Staffing Hiring Needs 260 6.4 Staff Task Requirements 262 Further Reading 267 7 Process Improvement: Six Sigma Approach to Laboratory Improvement 269 7.1 Introduction 269 7.2 DMAIIC Tools 273 7.3 Defining the Project 274 7.4 Measuring Variables 279 7.5 Analyze Data for the Project 282 7.6 Innovate and Improve the Project 287 7.7 Controlling the Results of the Project 289 Appendix 7.A Raw Baseline Data 295 Appendix 7.B Raw Control Data 295 Further Reading 296 8 Staff Training and Competency for a Cytogenetics Laboratory 299 8.1 Technician (Nonlicensed/Certified Lab Personnel) Training and Competency 300 8.2 Technologist (Licensed/ASCP‐Certified) Training and Competency 303 8.3 General Supervisor/Manager Training and Competency 307 8.4 Cytogenetic Technical Supervisor/Director Training and Competency 310 Further Reading 316 9 Training Program for Cytogenetic and FISH Technologists 319 9.1 Training Program Overview and Objectives 320 9.2 Program Content 323 9.3 Practical Component 332 9.4 Lectures, Quizzes, and Assignments 335 9.5 Trainee Competency and Completion of the Program 335 9.6 Trainee Handbook 336 9.7 Logs, Competency Forms, and Evaluation Forms 344 Further Reading 356 10 Training Program for Molecular Genetic Technologists 357 10.1 Training Overview and Objectives 358 10.2 Program Content 361 10.3 Practical Component 367 10.4 Lectures, Quizzes, and Assignments 370 10.5 Trainee Competency and Completion of the Training Program 370 10.6 Trainee Handbook 371 10.7 Logs, Competency Forms, and Evaluations Forms 379 Further Reading 383 Section III: Standard Operating Procedures 385 11 General SOP Information by Test and Preanalytic Procedures 387 11.1 Definition of an SOP 387 11.2 Example Template for Writing an SOP 390 11.3 CAP and ACMG Guidelines for Writing SOPs 393 11.4 Preanalytic Procedures: Accessioning Specimens for all Specimen Types 396 Further Reading 402 12 Analytic Procedures: Chromosome Analysis 405 12.1 Peripheral Blood and Percutaneous Umbilical Blood Specimen for Constitutional Disorders 410 12.2 Amniotic Fluid Specimens 440 12.3 Chorionic Villus Sampling 479 12.4 Solid Tissue Samples: Tissue Biopsies and Products of Conception 505 12.5 Bone Marrow and Blood for Hematologic Malignancies 534 12.6 Lymph Nodes and Solid Tumors 565 12.7 Breakage Syndromes 592 Appendix 12.A Cytogenetics Blood Culture and Harvest Worksheet 597 Appendix 12.B Daily Harvest Log 598 Appendix 12.C Test Slide Banding Log 599 Appendix 12.D Batch Banding Log 600 Appendix 12.E Analysis Sheet 601 Appendix 12.F Prenatal Culture and Harvest Worksheet 602 Appendix 12.G Culture Failure Log 603 Appendix 12.H Amniotic Fluid Count Guidelines—for Normal and Extended Studies 604 Appendix 12.I Hematologic Culture Log 605 Appendix 12.J Specimen Setup—Hematologic Malignancies 606 Appendix 12.K Solid Tumor Culture Worksheet 607 Further Reading 608 13 Analytic Procedures: Fluorescence in situ Hybridization (FISH) Analysis 613 13.1 General Information 613 13.2 CAP and ACMG Guidelines for Performing FISH Analyses 617 13.3 Cell Sorting for Plasma Cell Disorders for FISH Analysis 619 13.4 General Procedure for Direct Labeling of FISH Probes 624 13.5 Prenatal Multicolor Probes 641 13.6 ToTelVysion™ Multicolor DNA Probe Mixtures 654 13.7 Multicolor: Telomere, Centromere, and Paint Probes (Cytocell) 666 13.8 Microscope Analysis for Metaphase Scoring 680 13.9 Microscope Analysis for Interphase Scoring 685 13.10 Formalin‐Fixed Paraffin‐Embedded Procedure for FISH Analysis 693 13.11 HER2/ERBB2 FISH Analysis 700 13.12 UroVysion (Vysis) Bladder Cancer FISH Analysis 711 Appendix 13.Aa Constitutional FISH Probes 722 Appendix 13.Ab Hematologic FISH Assays 723 Appendix 13.B Probe Chart: Panels of Probes 724 Appendix 13.C FISH Direct Harvest Log 725 Appendix 13.D Batch Hybridization Log 726 Appendix 13.E General FISH Probe Analysis Sheet 727 Appendix 13.F FISH Probe Analysis Sheet—AML Panel 728 Appendix 13.G FISH Probe Analysis Sheet—MDS Panel 729 Appendix 13.H FISH Probe Analysis Sheet—CLL Panel 730 Appendix 13.I FISH Probe Analysis Sheet—NHL Panel 731 Appendix 13.J FISH Probe Analysis Sheet—MM Panel 732 Appendix 13.K FISH Probe Analysis Sheet—ALL Panel 733 Appendix 13.L FISH Analysis Sheet—BCR/ABL/ASS Triple Fusion 734 Appendix 13.M HER2 Analysis Sheet 735 Appendix 13.N UroVysion Analysis Sheet 736 Further Reading 736 14 Analytic Procedures: Chromosomal Microarray Analysis (CMA) 739 14.1 Test Principle 739 14.2 Comparing Conventional Chromosome Analysis, FISH Analysis and Chromosomal Microarray Analysis 741 14.3 Interpretation 745 14.4 Procedure Overview 747 Further Reading 752 15 Postanalytic Procedures 755 15.1 Reviewing and Reporting Cases for Chromosome Analysis 756 15.2 Reviewing and Reporting Cases for FISH Analysis 762 15.3 Reviewing and Reporting Cases for Microarray Analysis 765 15.4 Procedure for Avoidance and Detection of Clerical Errors Post Reporting 772 15.5 Specimen, Material and Record Retention, and Specimen Disposal 774 Appendix 15.A Record of Results Correspondence Log 778 Further Reading 779 Glossary 781 Index 811

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