PEPTIDOMICS -- CONTENTS -- Preface -- Contributors -- PART I STUDIES OF NATURALLY OCCURRING PEPTIDES -- 1 Analysis of the Peptidomes of Amphibian Skin Granular Gland Secretions-An Integrated Functional Genomic Strategy -- 1.1 Introduction -- 1.2 Historical Perspective -- 1.3 Contemporary Methods of Sample Acquisition -- 1.4 The Integrated Functional Genomic Strategy -- 1.5 How then can All Three Approaches be Integrated? -- 1.6 Limitations of Each Approach -- 1.7 Closing Thoughts -- 2 A Short History of Insect (Neuro)Peptidomics-A Personal Story of the Birth and Youth of an Excellent Model For Studying Peptidome Biology -- 2.1 Introduction -- 2.2 History -- 2.3 Present (and Future): Novel Technologies, New Data. . . -- 2.3.1 MALDI and (Nano)ESI TOFs and QTOFs -- 2.3.2 Peptide Displays -- 2.3.3 Ion Traps and FT-ICRs: Greater Variety of Instruments, Greater Variety of Sequences. . . -- 2.3.4 Orbitrap -- 2.3.5 FT-ICRs -- 2.3.6 Combining Peptide Chemical Analysis with Histological Localization: Peptidome Mass Spectrometry Imaging -- 2.4 Concluding Remarks -- 3 Peptidomics of Short Linear Cytolytic Peptides from Spider Venom -- 3.1 Introduction -- 3.2 Peptidomics: Identifying and Sequencing Novel AMPs -- 3.3 Genomics: Unraveling the Biosynthetic Pathway -- 3.4 Venomics: AMPs in the Spider Venom -- 3.4.1 Structural Features -- 3.4.2 Functional Features -- 3.4.3 Venomic Significance -- 4 Molecular Cloning Approaches to Peptidomics: The Identification of Novel cDNAs Encoding Neurotoxin-like Peptide Pools -- 4.1 Introduction -- 4.2 Spider Toxins-Combinatorial Peptide Libraries -- 4.3 EST Cloning Approach to Cloning Peptide Families -- 4.4 PCR-Based Amplification and Cloning Approaches -- 4.5 The Design and Use of Hybrid Primers for the Amplification of cDNAs Coding for Large Toxin-like Peptide Pools.

4.5.1 Multiple Alignment of the Existing Toxins and Toxin-like Sequences -- 4.5.2 The Design of Hybrid Partially Degenerate Primers -- 4.5.2.1 The Design of the 3 ́Region -- 4.5.2.2 The Design of the 5 ́Region and the Middle Part of the Primers -- 4.5.2.3 The Overall Length and Degeneracy -- 4.5.2.4 Annealing Temperature Matching -- 4.5.3 Optimization of the Amplification Conditions -- 4.5.4 Other Experimental Details -- 4.5.5 High Throughout Amplification and Cloning -- 4.5.6 Sequences Identification and Analysis -- 4.6 Polypeptide Toxins-Novel Applications in Drug and Pesticide Development -- 4.6.1 Analgesic Drugs -- 4.6.2 Insecticides -- 4.7 Concluding Remarks -- 4.7.1 RACE-PCR Issues -- 4.7.2 Sequence Quality Issues -- 4.7.3 Conclusion -- 5 Wheat Antimicrobial Peptides -- 5.1 Introduction -- 5.2 Materials and Methods -- 5.2.1 Isolation of AMPs -- 5.2.2 Reduction and Alkylation of Peptides [35] -- 5.2.3 MALDI-TOF MS -- 5.2.4 High-Resolution Two-Dimensional Gel Electrophoresis -- 5.3 Isolation of AMPs from T. kiharae Seeds -- 5.4 Sequence Determination of AMPs from T. kiharae Seeds -- 5.5 Comparative Analysis of D Defensins from Triticum and Aegilops Species -- 5.6 Discussion -- 5.7 Concluding Remarks -- 6 Immunopeptidomics: Applications to Dissect Immune Responses Through Proteomic-based Approaches -- 6.1 Introduction -- 6.1.1 Innate Immune Responses -- 6.1.2 Adaptive Immune Responses -- 6.2 Naturally Occurring Peptides and MHC-I Molecules: Role in Immune Surveillance and Tolerance -- 6.2.1 Structure and Basic Function of MHC-I Molecules -- 6.2.2 Relationship Between Naturally Occurring Peptides and MHC-I Function: A Peptidomics Approach -- 6.2.2.1 Peptide Analogs as Useful Tools to Dissect Immunological Mechanisms -- 6.2.2.2 Synthetic Peptide Libraries -- 6.3 Presentation of Naturally Processed Peptide Antigens by MHC-II Molecules.

6.3.1 Peptidomics Characterization of MHC-II-Binding Naturally Occurring Peptides -- 6.3.2 Functional Relationship Between MHC-II Molecules and Naturally Binding Peptides -- 6.4 Current Methodologies Applied to Peptidomics Analysis and the Identification of Naturally Processed Peptides -- 6.5 Potential Clinical Applications of Immunopeptidomic Analyses -- 7 Strategies for Reliable and Improved Identification of Peptides -- 7.1 Introduction -- 7.2 Neuropeptides and Hormones -- 7.3 Identification of Neuropeptides -- 7.4 Improved Identification of Peptides Using SwePep -- 7.5 Differential Display of Peptides -- 7.6 Differential Display of Peptides in Models of Parkinson's Disease -- PART II PEPTIDOMICS METHODS AND NEW DEVELOPMENTS -- 8 Peptidomics Approach to Proteomics -- 8.1 Introduction -- 8.2 Peptidomics Relating to Proteomics -- 8.2.1 Definition of Peptides Versus Proteins -- 8.2.2 Proteases-Producers of Peptides -- 8.3 Proteomics/Peptidomics -- 8.3.1 Background on Proteomics -- 8.3.2 General Objectives of Proteomics -- 8.3.3 Peptidomics -- 8.4 The 'omics Technologies -- 8.4.1 General Remarks -- 8.4.2 Sample Preparation -- 8.4.3 Basics of Mass Spectrometry -- 8.4.4 Bottom-Up Versus Top-Down Approaches -- 8.4.5 Separation Technologies Coupled MS -- 8.4.5.1 Two-Dimensional Gel Electrophoresis Followed by Mass Spectrometry (2DE-MS) -- 8.4.5.2 Liquid Chromatography Coupled to Mass Spectrometry (LC-MS) -- 8.4.5.3 Surface-Enhanced Laser Desorption/Ionization Coupled to Mass Spectrometry (SELDI-MS) -- 8.4.5.4 Capillary Electrophoresis Coupled to Mass Spectrometry (CE-MS) -- 8.4.6 Protein Arrays -- 8.5 Peptidomics Approach to Proteomics -- 9 The Importance of Sample Handling in Neuropeptidomics -- 9.1 Introduction -- 9.2 Issues Using Postmortem Brain Tissue -- 9.3 Enzyme Inactivation Methods -- 9.3.1 Denaturation -- 9.3.2 Thermal Denaturation.

9.3.3 Proteomics Denaturation Methods -- 9.3.4 Other Inactivation Methods -- 9.3.5 Protease Inhibitors -- 9.4 Brain Sample Preparation -- 9.5 Reliable Identification of Peptides in the Brain -- 9.6 Postmortem Time Effects on Peptides and Proteins -- 9.7 Protein Fragments as Degradation Markers -- 10 Affinity Peptidomics Approach to Protein Detection, Quantification, and Protein Affinity Assays: Application to Forensics and Biometrics -- 10.1 Introduction -- 10.1.1 Protein and Peptides: Separation, Staining, and Detection -- 10.1.2 Affinity-Based Assays and Hardware Realizations -- 10.1.3 Micro-/Macroarray-Based Protein Assays -- 10.2 Peptidomics Approach to Affinity Assays -- 10.3 Forensic and Biometrics Marker Proteins and Peptide Selection -- 10.4 Protein Markers and Peptide Selection -- 10.4.1 Myoglobin (MG) -- 10.4.2 Tumor Necrosis Factor-Alpha (TNF-α) -- 10.4.3 Fibrinogen (Fb)/D-Dimer -- 10.4.4 C-Reactive Protein (CRP) -- 10.4.5 Interferon(s) (IFN) -- 10.4.6 Interleukin(s) (IL) -- 10.4.6.1 IL-1 -- 10.4.6.2 IL-2 -- 10.4.6.3 IL-4 -- 10.4.6.4 IL-6 -- 10.4.6.5 IL-8 -- 10.4.6.6 IL-10 -- 10.4.7 Beta-Enolase (βE) -- 10.4.8 E-Selectin (ES) -- 10.4.9 Fibronectin (FN) -- 10.4.10 Haptoglobin (HG) -- 10.4.11 Heat Shock Protein(s) (HSP) -- 10.4.12 Insulin-like Growth Factor-1 (IGF-1) -- 10.4.13 Leptin (Lp) -- 10.4.14 Prolactin (Prl) -- 10.4.15 Transforming Growth Factor-Beta (TGF-β) -- 10.4.16 Adiponectin (Adn) -- 10.4.17 Insulin (In) -- 10.4.18 Matrix Metalloproteinases (MMPs 1, 2, 3, 9, 11) -- 10.4.19 Prostate-Specific Antigen (PSA) -- 10.4.20 Serum Amyloid-A (SAA) -- 10.4.21 Sex-Hormone-Binding Globulin (SHBG) -- 10.4.22 Surfactant-Associated Protein A (SP-A) -- 10.5 Concluding Remarks -- 11 Selective Depletion and Enrichment Methods for the Analysis of Protein and Peptide Pools -- 11.1 Introduction.

11.2 Peptide Mixture Refinement-General Approach to Combinatorial Peptidomics -- 11.3 Chemistry and Materials for Selective Reaction of Unmodified Amino Acid Side Chains -- 11.4 Selective Enrichment of Posttranslationally Modified Amino Acids -- 11.4.1 Phosphorylated Amino Acids -- 11.4.2 Selective Collection of Glycosylated Peptides -- 11.4.2.1 Phenyl Boronate Resins -- 11.4.2.2 Lectins -- 11.4.3 Amino Acids Modified by Covalent Attachment of Lipid Groups -- 11.5 Concluding Remarks -- 12 Detection of Target Peptides in Foods and Feeds by Mass Spectrometry -- 12.1 General Introduction -- 12.2 Introduction to GM Crops -- 12.3 Food Safety and Consumers Concerns -- 12.4 Current Detection Techniques of GM Material -- 12.5 Feed Contamination with Animal Protein in the Context of BSE and Control Measures -- 12.6 Plant Proteomics -- 12.7 Food Proteomics -- 12.8 Proteomic Investigations for the Detection of GM Crops -- 12.9 Case Study: Herbicide-Resistant GM Crops -- 12.9.1 Identification of CP4 EPSPS Peptides as Markers for Herbicide-Resistant GM Maize -- 12.9.2 Differential Protein Expression in GM and Non-GM Soya -- 12.10 Case Study: Gelatine Peptides as Markers for Contamination with Animal Protein -- 12.11 Concluding Remarks -- 13 Quantification of Polypeptides by Mass Spectrometry -- 13.1 Introduction -- 13.2 Development of Quantitative Mass Spectrometry for Polypeptide Analysis -- 13.3 Absolute Quantification of Polypeptides by Mass Spectrometry -- 13.3.1 The External Standard Method -- 13.3.2 The Internal Standard Method -- 13.4 Relative Quantification of Polypeptides by Mass Spectrometry -- 13.4.1 Methods Based on Derivatization -- 13.4.2 Methods Based on Metabolic Labeling -- 13.4.3 Label-Free Methods -- 13.5 Conclusions and Perspectives -- PART III PEPTIDOMICS APPROACH TO BIOMARKER DISCOVERY -- 14 Biomarker Discovery -- 14.1 Introduction.

14.2 Definition of Biomarkers.