[Methods in Molecular Biology] Capillary Electrophoresis of Proteins and Peptides Volume 1466 ||

This book provides a comprehensive survey of recent developments and applications of high performance capillary electrophoresis in the field of protein and peptide analysis with a distinct focus on the analysis of intact proteins. With practical detail, the contents cover different modes of capillary electrophoresis (CE) useful for protein and peptide analysis, CZE, CIEF, ACE, CGE, and different types of application such as the quality control of therapeutic proteins and monoclonal antibodies, clinical analyses of chemokines in tissues, qualitative and quantitative analysis of vaccine proteins, and determination of binding constants in complexes involving peptides or proteins. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and exhaustive, Capillary Electrophoresis of Proteins and Peptides: Methods and Protocols serves both beginners and experts with a collection of the current and most active topics in this vital field of study. Preface Contents Contributors Chapter 1: Chemical and Instrumental Approaches for Capillary Electrophoresis (CE)–Fluorescence Analysis of Proteins 1 Introduction 1.1 Labeling Reagents 1.2 Labeling Modes 1.3 Instrumentation 2 Materials 2.1 FQ Labeling of IgG 2.2 Operational Qualification Test of CE System 2.3 Sensitivity Checking 3 Methods 3.1 FQ Labeling of IgG 3.2 CE Method 3.2.1 Conditioning of a New Capillary 3.2.2 Operational Qualification Test of the CE System 3.2.3 Separation Method 3.3 Sensitivity Checking with Trypsin Inhibitor 3.4 Some Impurities Identification in IgG 4 Notes References Chapter 2: Discrimination of Glycoproteins from Unglycosylated Proteins in Capillary Electrophoresis: Two-Color LIF Detection Coupled with Post-column Derivatization 1 Introduction 2 Materials 2.1 Preparation of Solutions 2.2 Sample Preparation 2.3 Post-column Reactor 2.4 LIF Detector 3 Methods 4 Notes References Chapter 3: Capillary Zone Electrophoresis–Mass Spectrometry of Intact Proteins 1 Introduction 2 Materials 2.1 Instrumentation 2.2 Chemicals and Solutions 2.3 Proteins 3 Methods 3.1 Capillary Conditioning 3.2 Capillary Coating 3.2.1 PB-PVS Coating 3.2.2 PB-DS-PB Coating 3.2.3 Polyethyleneimine Silane (PEI) 3.3 CE-MS Analysis 3.3.1 Analysis of rhGH by Sheath-Liquid CE-MS Using PB-PVS Coated Capillaries 3.3.2 Analysis of Recombinant Human Interferon-­β-­1a by Sheath-­Liquid CE-MS Using PB-DS-PB-­Coated Capillaries 3.3.3 Analysis of Drug–Protein Conjugates by Sheathless CE-MS Using PEI-Coated Capillaries 3.3.4 Analysis of rhEPO by Sheathless CE-MS Using Neutrally Coated Capillaries 3.4 Data Analysis Using Maximum Entropy Processing 3.5 Concluding Remarks 4 Notes References Chapter 4: Screening of Small Intact Proteins by Capillary Electrophoresis Electrospray Ionization-Mass Spectrometry (CE-ESI-MS) 1 Introduction 2 Materials 2.1 Chemicals and Solutions 2.2 Instrumentation 2.2.1 CE 2.2.2 Coupling 2.2.3 Mass Spectrometry 3 Methods 3.1 Sample Treatment 3.2 Capillary Zone Electrophoresis 3.3 CE-MS Coupling 3.4 Mass Spectrometry 3.5 Validation 4 Notes References Chapter 5: Online Capillary IsoElectric Focusing-ElectroSpray Ionization Mass Spectrometry (CIEF-ESI MS) in Glycerol–Water Media for the Separation and Characterization of Hydrophilic and Hydrophobic Proteins 1 Introduction 2 Materials 2.1 Protein–Ampholyte Mixture 2.2 Anolyte and Catholyte Composition (See Note 7) 2.3 Capillary Preparation and Storage 2.4 Sheath Liquid Composition 3 Methods 3.1 CIEF Protocols (See Note 13) 3.2 Electrospray Interface (ESI) 3.3 Electrospray Interface Parameters 3.4 Data Treatment and Calibration Curve (See Fig. 2) 4 Notes References Chapter 6: On-Line Solid-Phase Extraction Capillary Electrophoresis Mass Spectrometry for Preconcentration and Clean-Up of Peptides and Proteins 1 Introduction 1.1 Solutions for the Sensitivity Issue in Capillary Electrophoresis 1.2 Coupling Solid-Phase Extraction to Capillary Electrophoresis 1.3 On-Line Solid- 2 Materials 3 Methods 3.1 Plasma Sample Pretreatment 3.2 SPE-CE-MS 3.2.1 Fused Silica Capillary Preparation 3.2.2 Construction of a Double-Frit Microcartridge 3.2.3 Preconcentration and Clean-Up 4 Notes References Chapter 7: Affinity Monolith-Integrated Microchips for Protein Purification and Concentration 1 Introduction 2 Materials 2.1 Microchip Fabrication 2.2 Monolith Fabrication 2.3 Antibody Immobilization Components 2.4 Protein Purification/Separation 3 Methods 3.1 Silicon Template Fabrication 3.2 Poly(Methyl Methacrylate) Microchip Fabrication 3.3 Photopatterning of Monoliths in Microchips 3.4 Immobilization of Anti-FITC 3.5 Protein Purification/Separation 4 Notes References Chapter 8: Analysis of Somatropin by Double-Injection Capillary-Zone Electrophoresis in Polybrene/Chondroitin Sulfate A Double-­Coated Capillaries 1 Introduction 1.1 Capillary Coating 1.1.1 Dynamic Adsorbed Capillary Coating 1.1.2 Static Coating of Fused Silica Capillaries Static Physically Adsorbed Coatings Static Covalent Capillary Coatings 1.2 Double-Injection Capillary Electrophoresis 2 Materials 2.1 Sample Preparation 2.2 CZE Running Protocol 3 Methods 3.1 Capillary Conditioning and Coating 3.2 Double-Injection Capillary Electrophoresis 3.3 Purity Determination 3.4 Result Treatment 4 Notes References Chapter 9: Poly(N,N-Dimethylacrylamide)-Based Coatings to Modulate Electroosmotic Flow and Capillary Surface Properties for Protein Analysis 1 Introduction 2 Materials 2.1 Synthesis of Poly(DMA-­NAS-­MAPS) and Poly(DMA-­GMA-­MAPS) 2.2 Coating of Fused Silica Capillaries with Poly (DMA-NAS-­MAPS) and Poly (DMA-­GMA- MAPS) 2.3 Blocking of Poly (DMA-NAS-­MAPS) Functional Groups (See Note 6) 2.4 Electroosmotic Flow Measurement 2.5 Alkaline Protein Separation 2.6 Acidic Protein Separation 3 Methods 3.1 Synthesis of Poly (DMA-NAS-­MAPS) and Poly (DMA-­GMA- MAPS) 3.2 Coating of Fused Silica Capillaries with Poly (DMA-NAS-­MAPS) and Poly (DMA-­GMA- MAPS) 3.3 Blocking of Poly (DMA-NAS-­MAPS) Functional Groups 3.4 Electroosmotic Flow Measurement 3.5 Alkaline Protein Separation 3.6 Acidic Protein Separation 4 Notes References Chapter 10: Measurement of Inflammatory Chemokines in Micro-­dissected Tissue Biopsy Samples by Chip-Based Immunoaffinity Capillary Electrophoresis 1 Introduction 2 Materials 2.1 Buffers and Solutions 2.2 Reagents 2.3 Patient and Control Samples 2.4 Instruments and Equipment 3 Methods 3.1 Biotinylation of the Anti-­chemokine Antibodies 3.2 Preparation of the Immunoaffinity Disks 3.3 Micro-dissection of Tissue Samples 3.4 ICE Measurement of Chemokines in Tissue Sections 3.5 Calculating Chemokine Values 4 Notes References Chapter 11: Separation of Recombinant Therapeutic Proteins Using Capillary Gel Electrophoresis and Capillary Isoelectric Focusing 1 Introduction 2 Materials 2.1 Instrumentation and Equipment 2.2 Solutions and Standards 3 Methods 3.1 Capillary Gel Electrophoresis Method 1 [16] 3.2 Capillary Gel Electrophoresis Method 2 [17] 3.3 Capillary Isoelectric Focusing (cIEF) [13] 4 Notes References Chapter 12: Characterization of Chemical and Physical Modifications of Human Serum Albumin by Capillary Zone Electrophoresis 1 Introduction 1.1 Chemical and Physical Modifications in Therapeutic Proteins 1.1.1 Physical Modifications 1.1.2 Chemical Modifications 1.2 Chemical and Physical Modifications in HSA Preparations 2 Materials 2.1 Instrument and Capillary 2.2 Preparation of PEO Solution at 2.22 mg/mL for Capillary Coating 2.3 Preparation of Buffers for CE Analyses 2.3.1 Buffer Used for EOF Measurement: 20 mM Sodium Phosphate pH 7.4 2.3.2 Buffer Used for HSA Analyses: 50 mM HEPES, 0.5 mM SDS, pH 7.5 10 mM SDS Solution 50 mM HEPES Solution with 0.5 mM SDS, pH 7.5 2.4 Sample Preparations 2.4.1 Neutral Marker Solution: 1 g/L of Thiourea (See Note 3) 2.4.2 HSA Solutions 0.05 M Solution of Copper Nitrate 1 mg/mL HSA with 30 μM of Copper Nitrate Freshly Prepared Each Day (See Note 6) 3 CZE Method 3.1 Coating Procedure 3.2 Inter-day PEO Regeneration 3.3 EOF Measurement Before Coating 3.4 EOF Measurement After Coating 3.5 HSA Analysis 4 Notes References Chapter 13: Capillary Electrophoresis Method for the Assessment of Erythropoiesis-Stimulating Agents in Final Formulations 1 Introduction 1.1 Overview on the Use of Capillary Electrophoresis for Intact Glycoproteins 1.1.1 Capillary Isoelectric Focusing (CIEF) 1.1.2 Capillary Electrophoresis: Sodium Dodecylsulfate (CE-SDS) 1.1.3 Capillary Zone Electrophoresis (CZE) 1.2 Analysis of Drug Products 2 Materials 2.1 Instrumentation and Setup 2.2 Background Electrolyte (BGE): 200 mM NaH2PO4, 1 mM NiCl2, pH 4.0 2.3 Sample Preparation 3 Methods 3.1 Capillary Cleaning, Conditioning, and Storage 3.1.1 Capillary Cleaning 3.1.2 Capillary Conditioning 3.1.3 Capillary Storage 3.2 Analysis of ESA Samples 4 Notes References Chapter 14: Quality Control of Therapeutic Monoclonal Antibodies at the Hospital After Their Compounding and Before Their Administration to Patients 1 Introduction 2 Materials 2.1 Infusion Bags Containing Monoclonal Antibodies 2.2 Solution Preparation 3 Methods 3.1 Sample Preparation 3.2 Capillary Pretreatment 3.3 CZE Analysis 3.4 CE Data Analysis 4 Notes References Chapter 15: Capillary Electrophoresis-Ultraviolet-Mass Spectrometry (CE-UV-MS) for the Simultaneous Determination and Quantification of Insulin Formulations 1 Introduction 2 Materials 2.1 Apparatus and Material 2.2 Chemical, Reagent, and Sample Solutions 2.2.1 Solutions for Capillary Electrophoresis-­Ultraviolet-­Mass Spectrometry (CE-UV-MS) 2.2.2 Solutions of Insulin Formulations for the Validation of the CE-UV-MS Procedure 2.2.3 Solutions of Insulin Formulations for the Routine Use of the CE-UV-MS Procedure 3 Methods 3.1 Capillary Electrophoresis- 3.2 Analysis of Insulin Formulations by CE-UV-MS: Validation of the Procedure 3.3 Analysis of Insulin Formulations by CE-UV-MS: Routine Use of the Procedure 4 Notes References Chapter 16: Applications of an Automated and Quantitative CE-Based Size and Charge Western Blot for Therapeutic Proteins and Vaccines 1 Introduction 2 Materials 2.1 Size-Based Western 2.1.1 Master Mix (2×) Reducing Solution Preparation (Final Volume 120 μL) (See Note 10) 2.1.2 Molecular Weight Standards Preparation (Final Volume 20 μL) (See Note 9) 2.1.3 Reducing Condition Sample Preparation 2.2 Charge-Based Western 2.2.1 Preparation of Premix 2.2.2 Preparation of Antibodies 2.2.3 Sample Preparation 3 Methods 3.1 Size-Based Western 3.2 Charge-Based Western 4 Notes References Chapter 17: Affinity Capillary Electrophoresis Applied to Investigation of Valinomycin Complexes with Ammonium and Alkali Metal Ions 1 Introduction 2 Materials and Instrumentation 2.1 Valinomycin Complexes with K+, Rb+, Cs+, and NH4+ Ions 2.1.1 Stock Solutions 2.1.2 Working Solutions 2.2 Valinomycin Complex with Na+ Ion 2.2.1 Stock Solutions 2.2.2 Working Solutions 2.3 Valinomycin Complex with Li+ Ion 2.3.1 Stock Solutions 2.3.2 Working Solutions 2.4 Instrumentation 3 Methods 3.1 Preconditioning of the Capillary 3.2 Injection of the Sample Solution 3.3 ACE Analyses 3.4 Determination of Binding Constant of Val-Alkali Metal/Ammonium Ion Complexes 4 Notes References Index Chemical And Instrumental Approaches For Capillary Electrophoresis (ce)-fluorescence Analysis Of Proteins / Isabelle Le Potier ... [et Al.] -- Discrimination Of Glycoproteins From Unglycosylated Proteins In Capillary Electrophoresis : Two-color Lif Detection Coupled With Post-column Derivatization / Takashi Kaneta -- Capillary Zone Electrophoresis-mass Spectrometry Of Intact Proteins / Elena Domínguez-vega, Rob Haselberg, And Govert W. Somsen -- Screening Of Small Intact Proteins By Capillary Electrophoresis Electrospray Ionization-mass Spectrometry (ce-esi-ms) / Sabine Neuberger, Angelina Rafai, And Christian Neusüss. Online Capillary Isoelectric Focusing-electrospray Ionization Mass Spectrometry (cief-esi Ms) In Glycerol-water Media For The Separation And Characterization Of Hydrophilic And Hydrophobic Proteins /meriem Mokaddem, Fanny D'orlyé, And Anne Varenne -- On-line Solid-phase Extraction Capillary Electrophoresis Mass Spectrometry For Preconcentration And Clean-up Of Peptides And Proteins / Fernando Benavente ... [et Al.] -- Affinity Monolith-integrated Microchips For Protein Purification And Concentration / Changlu Gao ... [et Al.] -- Analysis Of Somatropin By Double-injection Capillary-zone Electrophoresis In Polybrene/chondroitin Sulfate A Double-coated Capillaries / Ahmad Amini. Poly(n,n-dimethylacrylamide)-based Coatings To Modulate Electroosmotic Flow And Capillary Surface Properties For Protein Analysis / Laura Sola, Marina Cretich, And Marcella Chiari -- Measurement Of Inflammatory Chemokines In Micro-dissected Tissue Biopsy Samples By Chip-based Immunoaffinity Capillary Electrophoresis / Terry M. Phillips ... [et Al.] -- Separation Of Recombinant Therapeutic Proteins Using Capillary Gel Electrophoresis And Capillary Isoelectric Focusing / Caitlyn A. G. De Jong ... [et Al.] -- Characterization Of Chemical And Physical Modifications Of Human Serum Albumin By Capillary Zone Electrophoresis / Anne-lise Marie, Nguyet Thuy Tran, And Myriam Taverna. Capillary Electrophoresis Method For The Assessment Of Erythropoiesis-stimulating Agents In Final Formulations / Michel Girard, Anita Kane, And Sylvie Boucher -- Quality Control Of Therapeutic Monoclonal Antibodies At The Hospital After Their Compounding And Before Their Administration To Patients / Emmanuel Jaccoulet, Claire Smadja, And Myriam Taverna -- Capillary Electrophoresis-ultraviolet-mass Spectrometry (ce-uv-ms) For The Simultaneous Determination And Quantification Of Insulin Formulations / Julie Schappler And Serge Rudaz -- Applications Of An Automated And Quantitative Ce-based Size And Charge Western Blot For Therapeutic Proteins And Vaccines / Richard R. Rustandi ... [et Al.]. Affinity Capillary Electrophoresis Applied To Investigation Of Valinomycin Complexes With Ammonium And Alkali Metal Ions / Sille Štěpánová And Václav Kašička. Edited By Nguyet Thuy Tran And Myriam Taverna. Includes Bibliographical References And Index.