Proteomic profiling : methods and protocols

Preface
Contents
Contributors
Chapter 1: How Modern Mass Spectrometry Can Solve Ancient Questions: A Multi-Omics Study of the Stomach Content of the Oldest ...
	1 Mass Spectrometry and the Revolution in Biological Discovery
	2 Archeology Meets Multi-Omics Science
	3 Oetzi Under the Multi-Omics Microscope
		3.1 Proteomics
		3.2 Lipidomics
		3.3 Metabolomics
		3.4 Glycomics
		3.5 Metallomics
	4 Concluding Remarks
	References
Chapter 2: Step-by-Step Sample Preparation of Proteins for Mass Spectrometric Analysis
	1 Introduction
	2 Materials
		2.1 High-Throughput In-Gel Digestion of Proteins
			2.1.1 Excision of Protein Spots/Bands from Gels
			2.1.2 Destaining of Gel Plugs
			2.1.3 Reduction, Alkylation, and Enzymatic Digestion
			2.1.4 Peptide Cleaning and Extraction
		2.2 Filter-Aided Sample Preparation (FASP)
		2.3 One-Step Guanidine Method for Protein Lysis and Digestion
		2.4 StageTip Peptide Cleaning and Desalting
		2.5 TMT (Tandem Mass Tag) Labeling of Peptides
	3 Methods
		3.1 HTP In-Gel Digestion of Proteins
			3.1.1 Excision of Protein Spots/Bands from Gels
			3.1.2 Destaining of Gel Plugs
			3.1.3 Reduction, Alkylation, and Enzymatic Digestion
			3.1.4 Peptide Cleaning and Extraction
		3.2 Filter-Aided Sample Preparation (FASP)
		3.3 One-Step ``Guanidine´´ Method for Protein Lysis and Digestion
		3.4 StageTip Peptide Cleaning and Desalting
		3.5 Modified TMT Labeling of Peptides
	4 Notes
	References
Chapter 3: Lab-on-a-Filter Techniques for Economical, Effective, and Flexible Proteome Analysis
	1 Introduction
	2 Materials
		2.1 Reagents and Equipment
		2.2 Suspension Trapping (STrap) Digestion-Specific Reagents
		2.3 Filter-Aided Sample Preparation (FASP) Digestion-Specific Reagents
		2.4 PVDF Membrane Digestion-Specific Reagents
	3 Methods
		3.1 Protein Preparation from Different Sources
		3.2 STrap Digestion
		3.3 FASP Digestion
		3.4 PVDF Membrane Digestion
		3.5 Performance Analysis
	4 Notes
	References
Chapter 4: Released N-Glycan Analysis for Biotherapeutic Development Using Liquid Chromatography and Mass Spectrometry
	1 Introduction
	2 Materials
		2.1 Sample Preparation
		2.2 LC-FLR/MS Analysis
	3 Methods
		3.1 Sample Preparation
		3.2 LC-FLR-MS Analysis of Released N-Glycans
		3.3 Data Analysis
	4 Notes
	References
Chapter 5: Comprehensive Protocol to Simultaneously Study Protein Phosphorylation, Acetylation, and N-Linked Sialylated Glycos...
	1 Introduction
	2 Materials
		2.1 Sample Lysis, Reduction, Alkylation, and Digestion
		2.2 HLB Cartridge Peptide Purification
		2.3 TiO2 Batch Mode Enrichment
		2.4 Deglycosylation
		2.5 SIMAC Enrichment
		2.6 Second TiO2 Enrichment
		2.7 Acetyl Lysine IP
		2.8 Desalting Peptide Mixtures on R3 RP Columns
		2.9 HILIC Fractionation
		2.10 LC-ESI-MS/MS Analysis
	3 Methods
		3.1 Sample Lysis, Reduction, Alkylation, and Digestion
		3.2 HLB Cartridge Peptide Purification (See Notes 5 and 6)
		3.3 TiO2 Batch Mode Enrichment
		3.4 Deglycosylation
		3.5 SIMAC Enrichment
		3.6 Second TiO2 Enrichment
		3.7 Acetyl Lysine IP
		3.8 Desalting Peptide Mixtures on R3 RP Columns
		3.9 HILIC Fractionation
		3.10 LC-ESI-MS/MS Analysis (See Note 18)
	4 Notes
	References
Chapter 6: Phos-Tag Fluorescent Gel Staining for the Quantitative Detection of His- and Asp-Phosphorylated Proteins
	1 Introduction
	2 Materials (See Note 1)
		2.1 Protein Samples
		2.2 Solutions for Phos-Tag Magenta Gel Staining
		2.3 Gel Staining and Detection Equipment
	3 Methods
	4 Notes
	References
Chapter 7: Polyubiquitin Profile in Down Syndrome and Alzheimer´s Disease Brain
	1 Introduction
	2 Materials
		2.1 Sample Preparation
		2.2 Isolation of Polyubiquitinated Proteins
		2.3 Isolation of Total Protein
		2.4 Two-Dimensional Electrophoresis
		2.5 Image Analysis
		2.6 Protein Digestion
		2.7 Peptide Extraction and Clean-Up
	3 Methods
		3.1 Sample Preparation
		3.2 Isolation of Polyubiquitinated Proteins
		3.3 Isolation of Total Proteins
		3.4 Two-Dimensional Electrophoresis
		3.5 Image Analysis
		3.6 Protein Digestion
		3.7 Peptide Extraction
	4 Notes
	References
Chapter 8: Platform Methods to Characterize the Charge Heterogeneity of Three Common Protein Therapeutics by Imaged Capillary ...
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Reagents
	3 Methods
		3.1 icIEF Analysis of a Trastuzumab Biosimilar
			3.1.1 CpB Treatment
			3.1.2 Sample Preparation and icIEF
		3.2 icIEF Analysis of rhEPO
			3.2.1 Sample Concentration
			3.2.2 Sample Preparation and icIEF
		3.3 icIEF Analysis of a Fusion Protein
			3.3.1 Sample Preparation
			3.3.2 icIEF Analysis
	4 Notes
	References
Chapter 9: A Protocol for Isolation, Purification, Characterization, and Functional Dissection of Exosomes
	1 Introduction
	2 Materials
		2.1 Small-Scale Exosome Production
		2.2 Large-Scale Exosome Production
		2.3 Protein Quantitation
			2.3.1 Micro BCA Assay
			2.3.2 Protein Staining Densitometry
		2.4 Ultracentrifugation Exosome Isolation
		2.5 OptiPrep Density Gradient Exosome Isolation
		2.6 Cushion-Based Separation of Exosomes
		2.7 EpCAM Immunoaffinity Capture (IAC) Exosome Isolation
		2.8 Western Blot Analysis
		2.9 Nanoparticle Tracking Analysis (NTA)
		2.10 Aldehyde/Sulfate Latex (ALS) Bead-Based Capture
		2.11 Electron Microscopy (EM)
			2.11.1 Transmission EM
			2.11.2 Cryo EM
		2.12 Proteomics: Sample Preparation
			2.12.1 In-Solution Reduction, Alkylation, and Digestion
			2.12.2 StageTip Sample Cleanup
		2.13 Fluorometric Peptide Assay
		2.14 UHPLC-MS/MS
		2.15 Data Analysis
		2.16 Phenotypic Reprogramming of Cells by Exosomes: Dissecting Function
			2.16.1 Labeling Exosomes with Lipophilic Tracer
			2.16.2 Exosome Uptake
			2.16.3 Cell Activation Assay
			2.16.4 Transwell-Matrigel Invasion Assay
			2.16.5 Molecular Reprogramming of Cells by Exosomes
	3 Methods
		3.1 Small-Scale Exosome Production (2-20 μg)
		3.2 Large-Scale Exosome Production
		3.3 Protein Quantitation (See Note 7)
			3.3.1 Micro BCA Assay
			3.3.2 Protein Staining Densitometry
		3.4 Ultracentrifugation Exosome Isolation
		3.5 OptiPrep Density Gradient Exosome Isolation
		3.6 Cushion-Based Separation of Exosomes
			3.6.1 Cushion-Based Separation of Exosomes
			3.6.2 Ultracentrifugation Density Gradient Separation
		3.7 EpCAM Immunoaffinity Capture Exosome Isolation (See Note 15)
		3.8 Western Blot Analysis (See Note 16)
		3.9 Nanoparticle Tracking Analysis (NTA) (See Note 17)
		3.10 Aldehyde/Sulfate Latex Bead-Based Capture
		3.11 Electron Microscopy (EM) (See Note 18)
			3.11.1 Transmission EM
			3.11.2 CryoEM
		3.12 Proteomics: Sample Preparation
			3.12.1 In-Solution Reduction, Alkylation, and Digestion (See Note 19)
			3.12.2 StageTip Cleanup (See Note 23)
		3.13 Fluorometric Peptide Assay (See Note 25)
		3.14 UHPLC-MS/MS
		3.15 Data Analysis
		3.16 Phenotypic Reprogramming of Cells by Exosomes: Dissecting Function
			3.16.1 Labeling Exosomes with Lipophilic Tracer (See Note 27)
			3.16.2 Exosome Uptake (See Note 29)
			3.16.3 Cell Activation Assay
			3.16.4 Invasion Assay
			3.16.5 Molecular Reprogramming of Cells by Exosomes
	4 Notes
	References
Chapter 10: Human Plasma Extracellular Vesicle Isolation and Proteomic Characterization for the Optimization of Liquid Biopsy ...
	1 Introduction
	2 Materials
		2.1 PBPL Isolation
		2.2 Large EVs Isolation
		2.3 Small EVs Isolation
			2.3.1 CD63 Exo-FLOW Capture Kit
			2.3.2 ExoQuick ULTRA Kit
			2.3.3 exoEasy Kit
			2.3.4 Purification Mini Kit
		2.4 Albumin Depletion (See Note 14)
		2.5 EV Lysate Preparation
		2.6 Protein Quantitation (See Note 19)
		2.7 Western Blot Analysis
		2.8 Nanoparticle Tracking Analysis
		2.9 Transmission Electron Microscopy
		2.10 Proteomics: Sample Preparation
			2.10.1 In-Solution Reduction, Alkylation, and Digestion
			2.10.2 StageTip Sample Cleanup
		2.11 Fluorometric Peptide Assay
		2.12 UHPLC-MS/MS
		2.13 Data Analysis
	3 Methods
		3.1 PBPL Isolation
		3.2 Large EVs Isolation (See Note 22)
		3.3 Small EVs Isolation
			3.3.1 CD63 Exo-FLOW Capture Kit
			3.3.2 ExoQuick ULTRA Kit
			3.3.3 exoEasy Kit
			3.3.4 Purification Mini Kit
		3.4 Albumin Depletion (See Notes 14, 15, and 29)
		3.5 EV Lysate Preparation (See Notes 16-18)
		3.6 Protein Quantitation (See Note 19)
		3.7 Western Blot Analysis (See Notes 32-35)
		3.8 Nanoparticle Tracking Analysis (See Notes 32 and 36)
			3.8.1 NanoSight NS300 System
			3.8.2 ZetaView PMX-120 (See Notes 32 and 36)
		3.9 Transmission Electron Microscopy (See Notes 27, 37-39)
		3.10 Proteomics: Sample Preparation
			3.10.1 In-Solution Reduction, Alkylation, and Digestion
			3.10.2 StageTip Cleanup
		3.11 Fluorometric Peptide Assay (See Note 45)
		3.12 UHPLC-MS/MS
		3.13 Data Analysis
	4 Notes
	References
Chapter 11: Isolation of Extracellular Vesicles for Proteomic Profiling
	1 Introduction
	2 Materials
		2.1 Cell Culture with Serum-Free Culture Media
		2.2 Cell Culture with EV-Depleted Serum-Containing Culture Media
		2.3 Density Gradient Ultracentrifugation
		2.4 Methanol/Chloroform Precipitation
		2.5 In-solution Protein Digestion
		2.6 Desalting Using C18 Spin Column
	3 Methods
		3.1 Cell Culture with Serum-Free Culture Media
		3.2 Cell Culture with EV-Depleted Serum-Containing Culture Media
		3.3 Density Gradient Ultracentrifugation: Serum-Free Samples
		3.4 Density Gradient Ultracentrifugation: EV-Depleted Serum Samples
		3.5 Determination of Fraction Density
		3.6 Proteomic Profiling
			3.6.1 Methanol/Chloroform Precipitation
			3.6.2 In-solution Digestion of EVs
			3.6.3 Desalting Using C18 Spin Column (See Note 15)
	4 Notes
	References
Chapter 12: Isolation of Proteins from Extracellular Vesicles (EVs) for Mass Spectrometry-Based Proteomic Analyses
	1 Introduction
	2 Materials
	3 Methods
		3.1 EV-Cell Culture
		3.2 EV Isolation
		3.3 EV Lysis with Different Buffers
			3.3.1 Lysis with RIPA Buffer
			3.3.2 Lysis with Urea-Thiourea Solution
			3.3.3 Lysis with Guanidium-Hydrochloride (Gu-HCl) Solution
	4 Notes
	References
Chapter 13: Flow Cytometry as an Important Tool in Proteomic Profiling
	1 Introduction
	2 Materials
	3 Methods
		3.1 Panel Design
		3.2 Sample Preparation
		3.3 Red Cell Lysis
		3.4 Antibody and Viability Staining
		3.5 Data Analysis (See Fig. 4)
	4 Notes
	References
Chapter 14: Improved Immunoprecipitation to Mass Spectrometry Method for the Enrichment of Low-Abundant Protein Targets
	1 Introduction
	2 Materials
	3 Methods
		3.1 Biotinylation of Antibodies
		3.2 Immunoprecipitation with Streptavidin Magnetic Beads
		3.3 Enzyme Elution, Single-Pot Reduction Alkylation, and Mass Spectrometry Sample Preparation
		3.4 Second Elution for Antibody Epitope Mapping
	4 Notes
	References
Chapter 15: Multiplex Fluorescent Bead-Based Immunoassay for the Detection of Cytokines, Chemokines, and Growth Factors
	1 Introduction
	2 Materials
	3 Methods
		3.1 Assay Layout
		3.2 Serum Collection and Preparation
		3.3 Preparation of Standard Dilution Series (See Fig. 4)
		3.4 Preparation of Magnetic Beads and Samples (See Note 7)
		3.5 Preparation of Biotinylated Detection Antibodies (See Note 10)
		3.6 Preparation of Streptavidin- Phycoerythrin (SA-PE) (See Note 11)
		3.7 Main Assay Procedure
		3.8 Plate Reading
			3.8.1 Hardware Setup
			3.8.2 Adjustment of the Needle
			3.8.3 Start-Up
			3.8.4 Calibration
			3.8.5 Washing
			3.8.6 Protocol Setup
			3.8.7 Plate Reading
			3.8.8 Shutdown
			3.8.9 Data Analysis
	4 Notes
	References
Chapter 16: Bead-Based Multiplex Immunoassays: Procedures, Tips, and Tricks
	1 Introduction
	2 Technical Considerations
		2.1 Assay Format
		2.2 Sample Preparation and Storage
		2.3 Lab Materials and Equipment
		2.4 Assay Workflow
		2.5 Maintenance and Shutdown Procedures
		2.6 Data Analysis
			2.6.1 Bio-Plex Manager Software
			2.6.2 Bio-Plex Data Pro Software
	3 Concluding Remarks
	References
Chapter 17: Immunoaffinity-Based Liquid Chromatography Mass Spectrometric Assay to Accurately Quantify the Protein Concentrati...
	1 Introduction
	2 Materials
		2.1 Proteotypic Peptides
		2.2 Proteolysis
		2.3 Immunoprecipitation
		2.4 LC-MS/MS and Data Analysis
	3 Methods
		3.1 Peptide Preparation
		3.2 Proteolysis
		3.3 Immunoprecipitation
		3.4 LC-MS/MS-Based Peptide Quantification
		3.5 Data Analysis and Analyte Quantification
	4 Notes
	References
Chapter 18: Recombinant Anti-idiotypic Antibodies in Ligand Binding Assays for Antibody Drug Development
	1 Introduction
	2 Materials
	3 Methods
		3.1 PK Bridging Assay
		3.2 PK Assay Using Drug-Target Complex-Specific Antibodies
		3.3 ADA Bridging Assay
		3.4 Batch to Batch Consistency Test
		3.5 Accelerated Stability Test
	4 Notes
	References
Chapter 19: cDNA Display-Mediated Immuno-PCR (cD-IPCR): An Ultrasensitive Immunoassay for Biomolecular Detection
	1 Introduction
	2 Materials
		2.1 Synthesis of cnvK-rG Puromycin Linker (See Note 1) (Fig. 1a)
		2.2 Synthesis of BDA Coding cDNA Display Molecules
			2.2.1 In Vitro Transcription of DNA Coding for B Domain Protein A (BDA)
			2.2.2 UV Photocrosslinking Between mRNA and cnvK-rG Puromycin Linker
			2.2.3 In Vitro Translation (Synthesis of mRNA-BDA Fusion Molecule)
			2.2.4 Purification of mRNA-BDA Fusion Molecule
			2.2.5 Reverse Transcription to Form cDNA Display Molecules
			2.2.6 His-tag Affinity Purification of cDNA Display Molecule
		2.3 cDNA Display-Mediated Immune-PCR (cD-IPCR)
			2.3.1 Immobilization of Target Protein (Immunoglobulin G)
			2.3.2 Direct Type cD-IPCR
	3 Methods
		3.1 Synthesis of Puromycin Linker
			3.1.1 Reduction of Puromycin Segment
			3.1.2 EMCS Modification of Biotin cnvK Segment
			3.1.3 Cross-Linking of the Puromycin Segment and Biotin cnvK Segment
			3.1.4 HPLC Purification of Puromycin Linker
		3.2 Construction BDA DNA Templates
		3.3 Synthesis of cDNA Display Molecules
			3.3.1 In Vitro Transcription of BDA DNA
			3.3.2 Photocrosslinking Between BDA mRNA and cnvK-rG Puromycin Linker
			3.3.3 In Vitro Translation (Synthesis of mRNA-BDA Fusion Molecule)
			3.3.4 Immobilization of mRNA-BDA Fusion Molecule on Streptavidin Magnetic Beads
			3.3.5 Reverse Transcription to Form cDNA Display Molecules
			3.3.6 His-tag Affinity Purification of cDNA Display
		3.4 Direct cD-IPCR Detection of IgG
			3.4.1 Immobilization of IgG in Magnetic Beads
			3.4.2 Direct Type cD-IPCR
	4 Notes
	References
Chapter 20: Chromatin Immunoprecipitation (ChIP) to Study DNA-Protein Interactions
	1 Introduction
	2 Materials
		2.1 Nuclei Sample Preparation
		2.2 Prep Chromatin for IP
		2.3 Prep Antibody for IP
		2.4 Immunoprecipitation
	3 Methods
		3.1 Nuclei Sample Preparation
		3.2 Prep Chromatin for IP
		3.3 Prep Antibody for IP
		3.4 Immunoprecipitation
	4 Notes
	References
Chapter 21: Profiling Protein-DNA Interactions by Chromatin Immunoprecipitation in Arabidopsis
	1 Introduction
	2 Materials
		2.1 Consumable Materials and Reagents
		2.2 Equipment
	3 Methods
		3.1 Material Collection and Fixation
		3.2 Preparation of Antibody Beads
		3.3 Isolation of Nuclei, Chromatin Shearing, and ChIP
		3.4 Washing, Elution, Reverse Cross-Linking, and DNA Purification
		3.5 qPCR and Data Analyses
	4 Notes
	References
Chapter 22: Biotin Proximity Labeling for Protein-Protein Interaction Discovery: The BioID Method
	1 Introduction
	2 Materials
		2.1 Biotin Labeling Time Course Validation
		2.2 Biotinylated Protein Generation
		2.3 Cell Harvesting and Lysate Generation
		2.4 Biotinylated Protein Purification
	3 Methods
		3.1 Biotin Labeling Time Course Validation
		3.2 Biotinylation and Cell Harvest
		3.3 Cell Lysis
		3.4 Affinity Purification with Magnetic Streptavidin Beads
		3.5 Western Blot Visualization
		3.6 Data Analysis
	4 Notes
	References
Chapter 23: Studying OTUD6B-OTUB1 Protein-Protein Interaction by Low-Throughput GFP-Trap Assays and High-Throughput AlphaScree...
	1 Introduction
	2 Materials
		2.1 Protein Expression and Purification
		2.2 Immunoprecipitation Using GFP-Traps
		2.3 ``AlphaScreen´´ Homogeneous Proximity Assay
	3 Methods
		3.1 Protein Expression and Purification Protocols
			3.1.1 Recombinant Production and Purification of His-OTUB1
			3.1.2 Recombinant Production and Purification of GST-OTUD6B
		3.2 GFP-Trap Immunoprecipitation
			3.2.1 Seeding of Cells and Transfection
			3.2.2 Harvesting the Cells
			3.2.3 Bead Equilibration of GFP-Trap-Agarose
			3.2.4 GFP-Trap (Binding, Washing and Elution)
		3.3 ``AlphaScreen´´ Homogeneous Proximity Assay
			3.3.1 AlphaScreen Matrix Titration Experiment
			3.3.2 AlphaScreen PPI Assay at Defined Concentration
	4 Notes
	References
Chapter 24: Thermal Shift Assay for Exploring Interactions Between Fatty Acid-Binding Protein and Inhibitors
	1 Introduction
	2 Materials
		2.1 Protein Preparation and Purification
		2.2 Protein Melt Reaction Using Real-Time PCR
	3 Methods
		3.1 Protein Preparation and Purification
		3.2 Thermal Shift Assay
			3.2.1 Protein Thermal Shift Assay Mix Preparation (See Table 1)
			3.2.2 Set the Real-Time PCR Program (See Table 2)
			3.2.3 Data Analysis
	4 Notes
	References
Chapter 25: Isolation and Purification of Mitochondria from Cell Culture for Proteomic Analyses
	1 Introduction
	2 Materials
		2.1 Isolation of Crude Mitochondria
		2.2 Density Gradient
	3 Methods
		3.1 Isolation of Crude Mitochondria (See Note 1)
		3.2 Density Gradient Centrifugation (See Note 13)
	4 Notes
	References
Chapter 26: Investigating the Adipose Tissue Secretome: A Protocol to Generate High-Quality Samples Appropriate for Comprehens...
	1 Introduction
	2 Materials
		2.1 General Hardware and Consumables
		2.2 Tissue Biopsies and Isolation of Preadipocytes
		2.3 Differentiation of Preadipocytes
		2.4 Quality Control of Differentiation by Oil Red O Protocol
		2.5 Collecting Secreted Peptides/Proteins
	3 Methods
		3.1 Tissue Biopsies
		3.2 Isolation of Preadipocytes
		3.3 Differentiation of Preadipocytes
		3.4 Quality Control of Differentiation by Oil Red O Staining
		3.5 Collecting Secreted Peptides/Proteins
		3.6 Concentration of Supernatant for Proteomic Analyses
	4 Notes
	References
Chapter 27: Methods for Proteomics-Based Analysis of the Human Muscle Secretome Using an In Vitro Exercise Model
	1 Introduction
	2 Materials
		2.1 General Consumables
		2.2 Cell Culture
		2.3 Targeted Proteomics
		2.4 Untargeted Proteomics
	3 Methods
		3.1 Enrichment of CD56-Positive Myoblasts
		3.2 Fusion to Myotubes and Stimulation
		3.3 Collection of Medium for Targeted Secretome Studies
		3.4 Collection of Medium for Untargeted Proteomics
	4 Notes
	References
Chapter 28: Western Blotting Using In-Gel Protein Labeling as a Normalization Control: Advantages of Stain-Free Technology
	1 Introduction
	2 Materials
		2.1 Tissue Sample Preparation
		2.2 SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE)
		2.3 Protein Transfer
		2.4 Antibodies for Immunostaining
	3 Methods
		3.1 Sample Preparation
		3.2 SDS-Page
		3.3 Protein Transfer
		3.4 Immunostaining
	4 Notes
	References
Chapter 29: Technical Considerations for Contemporary Western Blot Techniques
	1 Introduction
	2 Immunodetection
		2.1 Blocking
		2.2 Wash Volume and Agitation
		2.3 Antibody Selection and Dilution
			2.3.1 Primary Immunodetection
			2.3.2 Secondary Immunodetection
			2.3.3 Antibody-Specific Ligands
	3 Image Acquisition
		3.1 Visualization Methods
			3.1.1 Chemiluminescence Detection
			3.1.2 Fluorescence Detection
		3.2 Stripping and Reprobing
		3.3 Imaging Systems
			3.3.1 Digital Imaging for Chemiluminescence Detection
			3.3.2 Digital Imaging for Fluorescence
		3.4 Sensitivity
		3.5 Scales of Reporting
			3.5.1 Log
			3.5.2 Orders of Magnitude/Decades
			3.5.3 Bit Depth and Bits
			3.5.4 Dynamic Range
			3.5.5 Resolution
	4 Image Analysis
		4.1 Molecular Weight (Size) Estimation
		4.2 Quantitation
			4.2.1 Total Protein Normalization
			4.2.2 Housekeeping/Single Protein Normalization
	References
Chapter 30: Simple Western: Bringing the Western Blot into the Twenty-First Century
	1 Introduction
	2 Materials
		2.1 Simple Western Size Reagents
		2.2 Simple Western Charge Reagents
	3 Methods
		3.1 Measuring Protein Expression with Protein Normalization
		3.2 Characterizing Charge Heterogeneity
	4 Notes
	References
Chapter 31: Development of Peptide Ligands for Targeted Capture of Host Cell Proteins from Cell Culture Production Harvests
	1 Introduction
	2 Materials
		2.1 Protein Mix Formulation for Library Screening
		2.2 Dual Fluorescence Library Screening
			2.2.1 Incubation of the OBOP Library
			2.2.2 Manual Bead Sorting
			2.2.3 ClonePix 2 Bead Sorting
		2.3 Confirmation of HCP Binding by Selected Peptides
		2.4 Proteomic Analysis of the Chromatographic Fractions
			2.4.1 Filter-Aided Sample Preparation (FASP)
			2.4.2 nanoLC-MS/MS Analysis
	3 Methods
		3.1 Protein Mix Formulation for Library Screening
		3.2 Dual Fluorescence Library Screening
			3.2.1 Incubation of the OBOP Library
			3.2.2 Manual Bead Sorting
			3.2.3 ClonePix 2 Bead Sorting
		3.3 Confirmation of HCP Binding by Selected Peptides
		3.4 Proteomic Analysis of the Chromatographic Fractions
			3.4.1 Filter-Aided Sample Preparation (FASP)
			3.4.2 nanoLC-MS/MS Analysis
			3.4.3 Statistical Analysis of the DDA Proteomics Data
	4 Notes
	References
Chapter 32: Sample Preparation of Secreted Mammalian Host Cell Proteins and Their Characterization by Two-Dimensional Electrop...
	1 Introduction
	2 Materials
		2.1 Sample Preparation and Protein Assay
		2.2 2D Electrophoresis: First Dimension IEF
		2.3 2D Electrophoresis: Equilibration and Second-Dimension SDS-PAGE
		2.4 Staining and Blotting
		2.5 Immunodetection and Analysis
		2.6 Image Analysis Software
	3 Methods
		3.1 Sample Preparation and Protein Assay
		3.2 2-D Electrophoresis: First Dimension IEF
		3.3 2D Electrophoresis: Equilibration and Second-Dimension SDS-PAGE
		3.4 Staining and Blotting
		3.5 Immunodetection
		3.6 Software Analysis and Generation of a Match Rate for Assessment of Antibody Coverage
	4 Notes
	References
Chapter 33: Quantitative Proteomic Analysis Using Formalin-Fixed, Paraffin-Embedded (FFPE) Human Cardiac Tissue
	1 Introduction
	2 Materials
		2.1 Sample Preparation and Protein Digestion
		2.2 LC-MS and Functional Analysis
	3 Methods
		3.1 Protein Extraction from FFPE Tissue
		3.2 Protein Digestion
		3.3 LC-MS/MS Analysis, Quantification, and Functional Analysis
		3.4 Quantitative Proteomics Using FFPE Cardiac Tissue: An Example
	4 Notes
	References
Chapter 34: Chloroplast Isolation and Enrichment of Low-Abundance Proteins by Affinity Chromatography for Identification in Co...
	1 Introduction
	2 Materials
		2.1 Chloroplast Isolation from Pea (Pisum sativum)
		2.2 Chloroplast Isolation from Arabidopsis
		2.3 Chloroplast Stroma Extraction
		2.4 Gel Filtration
		2.5 Ion Exchange Chromatography
		2.6 Hydrophobic Interaction Chromatography (HIC)
		2.7 ATP/Purvalanol B Affinity Chromatography
		2.8 Heat Treatment of Isolated Chloroplasts and Protein Extraction
		2.9 Eu3+-IDA Column Affinity Chromatography
	3 Methods (See Note 5)
		3.1 Chloroplast Isolation from Pea (See Note 6)
		3.2 Chloroplast Isolation from Arabidopsis (See Note 6)
		3.3 Estimation of the Chlorophyll Content
		3.4 Chloroplast Stroma Extraction
		3.5 Gel Filtration
		3.6 Ion Exchange Chromatography
		3.7 Hydrophobic Interaction Chromatography (HIC)
		3.8 ATP/Purvalanol B Affinity Chromatography
		3.9 Heat Treatment of Chloroplasts and Protein Extraction
		3.10 Eu3+-IDA Column Affinity Chromatography
	4 Notes
	References
Chapter 35: Principles of Protein Labeling Techniques
	1 Introduction
	2 Materials
	3 Methods
		3.1 Labeling with Fluorescent Dyes Prior to Electrophoretic Separations
		3.2 Protein Labeling with Stable Isotopes
	4 Notes
	References
Chapter 36: Mechanical/Physical Methods of Cell Disruption and Tissue Homogenization
	1 Introduction
	2 Bead Impact Methods: Shaking Vessel
		2.1 Theory
		2.2 Practical Aspects
	3 Bead Impact Methods: Stirred Agitated Beads
		3.1 Theory
		3.2 Practical Aspects
	4 Rotor-Stator Homogenizer
		4.1 Theory
		4.2 Practical Aspects
	5 Mortar and Pestle Tissue Grinders: Shear by Mechanical Pressure
	6 High Pressure Batch: Expanding Fluids
		6.1 Theory
			6.1.1 The French Press G-M
			6.1.2 The Parr Cell Disruption Vessel
		6.2 Practical Aspects
			6.2.1 French Press G-M
			6.2.2 PARR Cell Disruption Vessel
	7 HIGH Pressure Flow: Shear Through a Valve or Tube
		7.1 Theory
			7.1.1 High Pressure Valve with Impingement Wall: Gaulin and Rannie
			7.1.2 High Pressure Flow Narrow Tubes or Opposed Jets: Microfluidics
		7.2 Practical Aspects
			7.2.1 Practical Aspects Using High Pressure Valve with Impingement Wall: Gaulin and Rannie
			7.2.2 Practical Aspects: High Pressure Narrow Tubes/Opposed Jets
	8 Low Pressure: Shear by Droplet´s Impingement
	9 Ultrasonic Processors: Shear by Collapsing Bubbles
		9.1 Theory
		9.2 Practical Aspects
	10 Extraction Across an Electromotive Field
		10.1 Theory
		10.2 Practical Aspects
	11 Lysis with Pulsed Electric Fields
		11.1 Theory
		11.2 Practical Aspects
	12 Microwave-Assisted Centrifugation
		12.1 Theory
		12.2 Practical Aspects
	References
Index