Capillary Electrophoresis of Proteins and Peptides: Methods and Protocols

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