Mass Spectrometry of Glycoproteins: Methods and Protocols

Preface
Contents
Contributors
1. Glycosylation of Therapeutic Proteins: A Critical Quality Attribute • Arnaud Delobel
	1 Introduction
		1.1 Glycoproteins as Therapeutics
		1.2 Structure of Glycosylated Proteins
			1.2.1 Monosaccharides, the Building Blocks of Glycans
			1.2.2 N-Glycans
			1.2.3 O-Glycans
		1.3 Impact of the Expression System on the Glycosylation Profile
		1.4 Impact of the Manufacturing Process on Protein Glycosylation
		1.5 Biosimilar Drugs
	2 Impact of the Glycosylation Profile on the Properties of Therapeutic Proteins
		2.1 Influence of Glycosylation on Solubility and Stability of Glycoproteins
		2.2 Influence of Glycosylation on Pharmacokinetics
		2.3 Influence of Glycosylation on Receptor Binding
		2.4 Influence of Glycosylation on Immunogenicity
		2.5 Glycosylation of Monoclonal Antibodies (mAbs) and Related Constructs
	3 The Role of Mass Spectrometry on the Analysis of Glycosylation
	References
2. Characterization of Protein Glycoforms at Intact Level by Orbitrap Mass Spectrometry • Dan Bach Kristensen, Trine Meiborg Sloth, Martin Ørgaard, and Pernille Foged Jensen
	1 Introduction
	2 Materials
		2.1 LC-MS System
		2.2 MS Data Processing
		2.3 Columns
		2.4 Solvents and Solutions
			2.4.1 Native MS Solvents
			2.4.2 RP LC-MS Solvents
	3 Methods
		3.1 Native SEC MS
			3.1.1 Instrument Settings and Methods
			3.1.2 Start-Up (New Column)
			3.1.3 Start-Up (Used Column)
			3.1.4 Sample Analysis
			3.1.5 Column Storage and Cleaning
		3.2 Native CIEX MS
			3.2.1 Instrument Settings and Methods
			3.2.2 Start-Up (New Column)
			3.2.3 Start-Up (Used Column)
			3.2.4 Sample Analysis
			3.2.5 Column Storage (See Subheading 3.1.5)
		3.3 Intact RP LC-MS
			3.3.1 Instrument Settings and Methods
			3.3.2 Start-Up (New Column)
			3.3.3 Start-Up (Used Column)
			3.3.4 Sample Analysis
			3.3.5 Column Storage
		3.4 Data Processing
	4 Notes
	References
3. Analysis of Intact Glycoproteins by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry • Estela Giménez, Montserrat Mancera-Arteu, Fernando Benavente, and Victoria Sanz-Nebot
	1 Introduction
	2 Materials
		2.1 Instrumentation
		2.2 Chemicals and Solutions
	3 Methods
		3.1 Sample-Matrix Preparation Procedure
			3.1.1 Fast Evaporation Method
			3.1.2 Vacuum Drying Method
			3.1.3 ILM Method
		3.2 MALDI-TOF MS Analysis
	4 Notes
	References
4. Fc Glycosylation Characterization of Human Immunoglobulins G Using Immunocapture and LC-MS • Yosra Helali, Shilpee Sharma, Marie Vandeput, Dansala Welba, Pierre Van Antwerpen, Arnaud Marchant, and Cédric Delporte
	1 Introduction
	2 Materials
		2.1 Chemicals
		2.2 Equipment
	3 Methods
		3.1 Sample Preparation
			3.1.1 IgG Binding
			3.1.2 IDEZ Digestion
			3.1.3 Collecting the Fc Fraction
			3.1.4 IgG Denaturation
			3.1.5 Digestion
			3.1.6 Labeling
			3.1.7 Cleaning up with HILIC-SPE
			3.1.8 Prepare the Samples for the Injection
		3.2 Analysis Conditions
			3.2.1 LC Conditions
			3.2.2 ESI-Q-TOF and FLD Detector Parameters
			3.2.3 Data Analysis
		3.3 General Discussion on Data Interpretation in Disease
	4 Notes
	References
5. Fast Afucosylation Profiling of Glycoengineered Antibody Subunits by Middle-Up Mass Spectrometry • Elsa Wagner-Rousset, Olivier Colas, Stéphane Chenu, Yannis-Nicolas François, Davy Guillarme, Sarah Cianferani, Yury O. Tsybin, Jonathan Sjögren, Arnaud Delobel, and Alain Beck
	1 Introduction
		1.1 Characterization of Obinutuzumab Subunits Under Reducing Conditions and EndosS2 Digestion (Middle-Level, 23-54 kDa Fragmen...
		1.2 Characterization of Benralizumab Subunits After Enzymatic Cleavage (IdeS), Reduction and EndoS Deglycosylation (Middle-Lev...
		1.3 Characterization of mAb A (Produced in CHO Cells) and mAb B (Produced in CHO Cells) and Subunits Cultivated After Enzymati...
	2 Materials
		2.1 Chemicals
		2.2 mAbs
		2.3 Ultraperformance Liquid Chromatography
		2.4 Mass Analysis
		2.5 Reagents Preparation
	3 Methods
		3.1 General Principle
		3.2 Sample Preparation
			3.2.1 Reduction
			3.2.2 IdeS Digestion and Reduction
		3.3 Data Analysis
			3.3.1 MS Data Treatment
			3.3.2 UV Data Treatment
	4 Notes
	References
6. Characterization of Glycosylated Proteins at Subunit Level by HILIC/MS • Valentina D’Atri and Davy Guillarme
	1 Introduction
	2 Materials
		2.1 Generation of Protein Subunits
		2.2 Hydrophilic Interaction Chromatography (HILIC) Coupled to Electrospray Ionization Mass Spectrometry (ESI-MS)
	3 Methods
		3.1 Generation of Protein Subunits
		3.2 HILIC-MS Data Treatment and Assessment of N-Glycan Profile
	4 Notes
	References
7. Analysis of Monoclonal Antibody Glycopeptides by Capillary Electrophoresis-Mass Spectrometry Coupling (CE-MS) • Josiane Saadé, Michael Biacchi, Jérémie Giorgetti, Antony Lechner, Alain Beck, Emmanuelle Leize-Wagner, and Yannis-Nicolas François
	1 Introduction
	2 Materials
		2.1 Reagents and Buffers
		2.2 Instrumentation
	3 Methods
		3.1 Trypsin Digestion
		3.2 CE-ESI-MS Analysis
		3.3 MS and MS/MS Data Analysis
	4 Notes
	References
8. Enrichment of Intact Glycopeptides Using Strong Anion Exchange and Electrostatic Repulsion Hydrophilic Interaction Chromatography • Abel Bermudez and Sharon J. Pitteri
	1 Introduction
	2 Materials
		2.1 Sample Homogenization (See Note 1)
		2.2 Sample Digestion
		2.3 SAX-ERLIC
		2.4 Reversed-Phase Chromatography and Mass Spectrometry
		2.5 Instrumentation
	3 Methods
		3.1 Sample Homogenization
		3.2 Trypsin Digestion
		3.3 Strong Anion Exchange and Electrostatic Repulsion Hydrophilic Interaction Liquid Chromatography (SAX-ERLIC)
		3.4 Reverse-Phase Liquid Chromatography-Tandem Mass Spectrometry Analysis
		3.5 Glycoprotein Identification
	4 Notes
	References
9. In-Depth Glycan Characterization of Therapeutic Glycoproteins by Stepwise PGC SPE and LC-MS/MS • Myung Jin Oh, Youngsuk Seo, Unyong Kim, and Hyun Joo An
	1 Introduction
	2 Materials
		2.1 Samples
		2.2 Detergent Removal
		2.3 Release of N-Glycans
		2.4 Release of N-Glycans
		2.5 LC Separation and MS Analysis of Glycans
	3 Methods
		3.1 Detergent Removal
		3.2 Enzymatic Release of N-Glycans
		3.3 Purification and Fractionation of Neutral and Sialylated Glycans on mAb Using PGC  SPE
		3.4 Purification and Fractionation of Neutral, Sialylated, and Phosphorylated Glycans on Lysosomal Enzyme Using PGC  SPE
		3.5 PGC-LC/MS Analysis
		3.6 MS Data Analysis
		3.7 Glycan Profiling
	4 Notes
	References
10. Site-Specific N-glycosylation Analysis of Recombinant Proteins by LC/MSE • Kevin Canis, Estelle Garénaux, and Jean-François Boe
	1 Introduction
	2 Materials
		2.1 Equipment and Consumables
		2.2 Reagents
		2.3 Equipment
			2.3.1 Hardware
			2.3.2 Software
			2.3.3 Column
	3 Methods
		3.1 In Silico Analysis
			3.1.1 Amino Acid Sequence Examination
			3.1.2 Glycosylation Properties of the Expression System
			3.1.3 Creation of the Glycopeptide Database
		3.2 Sample Preparation and Analysis
			3.2.1 Sample Deformulation
			3.2.2 Sample Reduction
			3.2.3 Sample Alkylation
			3.2.4 Sample Desalting
			3.2.5 Proteolytic Digestion
			3.2.6 Glycopeptide Enrichment (optional)
			3.2.7 LC-MS Analysis
				Liquid Chromatography
				Mass Spectrometry
		3.3 Data Interpretation
			3.3.1 Detection of Glycopeptide-Related Clusters
			3.3.2 Identification of the Peptide Sequences
			3.3.3 Determination of the Glycan Repertoire
			3.3.4 Evaluation of Peptide´s Microheterogeneity
			3.3.5 Sample Monitoring/Batch-to-Batch Comparison
		3.4 Complementary MS-Based Approaches
	4 Notes
	References
11. Mapping O-glycosylation Sites Using OpeRATOR and LC-MS • Maria Nordgren, Andreas Nägeli, Helén Nyhlén, and Jonathan Sjögren
	1 Introduction
	2 Materials
		2.1 Protein Buffer Exchange
		2.2 O-Glycoprotein Digestion
		2.3 Sample Cleanup Using Graphite Spin Columns
		2.4 O-Glycopeptide Separation by Hydrophilic Interaction Chromatography and Tandem MS Analysis of O-Glycopeptides Using Collis...
	3 Methods
		3.1 Protein Buffer Exchange
		3.2 O-Glycoprotein Digestion
		3.3 Sample Cleanup Using Graphite Resin
		3.4 Hydrophilic Interaction Chromatography and Tandem MS Analysis of O-Glycopeptides Using Stepping Collision Energy CID Fragm...
	4 Notes
	References
12. Site-Specific O-Glycosylation Analysis by Liquid Chromatography-Mass Spectrometry with Electron-Transfer/Higher-Energy Collisional
	1 Introduction
	2 Materials
		2.1 Sample Preparation
		2.2 Liquid Chromatography-Mass Spectrometry and Database Search
	3 Methods
		3.1 Sample Preparation: Preparation of Tryptic Digests
		3.2 Liquid Chromatography-Mass Spectrometry
		3.3 Database Search Analysis
	4 Notes
	References
13. Profiling of N-Linked Oligosaccharides of a Glycoprotein by UPLC-FLR-ESI-MS After Derivatization with Fluorescent Anthranilamide • Claire I. Butré, Eric Largy, and Arnaud Delobel
	1 Introduction
	2 Materials
		2.1 Apparatus
		2.2 UPLC Preparation
		2.3 Sample Preparation
		2.4 Standard
	3 Methods
		3.1 Sample Preparation (see Note 2)
			3.1.1 N-Glycan Release-One-Step Protocol
			3.1.2 N-Glycan Release-Two-Step Protocol
			3.1.3 Derivatization (see Note 5)
			3.1.4 Purification
		3.2 UHPLC Separation
		3.3 MS Conditions
	4 Notes
	References
14. Evaluating N-Glycosylation of a Therapeutic Monoclonal Antibody Using UHPLC-FLR-MS with RapiFluor-MS Labeling • Rosie Upton, James Duffy, Sam Clawson, and David Firth
	1 Introduction
	2 Materials
		2.1 Sample Preparation
			2.1.1 Exoglycosidase Digestion (Optional)
		2.2 Mass Spectrometer Setup
	3 Methods
		3.1 Sample Denaturation and Deglycosylation
		3.2 Glycan Labeling
		3.3 SPE Cleanup
		3.4 Exoglycosidase Digestion (Optional)
		3.5 LC-FLR-MS Analysis
		3.6 Released N-Glycan Analysis
		3.7 Optional Exoglycosidase Digestion
	4 Notes
	References
15. Routine Analysis of N-Glycans Using Liquid Chromatography Coupled to Routine Mass Detection • Ximo Zhang, Vithiya Vimalraj, and Mitul Patel
	1 Introduction
	2 Material
		2.1 Material for Sample Preparation of Released N-Glycan
		2.2 LC-MS of Released N-Glycans
	3 Method
		3.1 Sample Preparation of Released N-Glycans
		3.2 Routine LC-MS Analysis of Released N-Glycans
			3.2.1 LC-FLR-MS Data Generation
			3.2.2 Data Analysis
		3.3 High-Throughput Release N-Glycan Analysis
		3.4 Applications of RapiFluor-MS N-Glycan Method
			3.4.1 Cell Line and Process Development
			3.4.2 Analytical Characterization
	4 Notes
	References
16. Online 2D-LC for Complex N-Glycan Analysis from Biopharmaceuticals • Sonja Schneider, Edgar Naegele, and Sonja Krieger
	1 Introduction
	2 Materials
		2.1 Sample Preparation
		2.2 (2D)-LC/MS Analysis
			2.2.1 Comprehensive 2D-LC HILIC/WAX Workflow
			2.2.2 Multiple Heart-Cutting 2D-LC WAX/HILIC Workflow
			2.2.3 LC Columns
			2.2.4 Apparatus and Software
	3 Methods
		3.1 Protein Deglycosylation
		3.2 Glycan Labeling (GlycoProfile 2-AB Labeling Kit)
		3.3 Glycan Purification
			3.3.1 Glycan Adsorption
			3.3.2 Glycan Elution
		3.4 2D-LC/MS Analysis
			3.4.1 Comprehensive 2D-LC Analysis HILIC/WAX
			3.4.2 Multiple Heart-Cutting 2D-LC Analysis WAX/HILIC
	4 Notes
	References
17. Profiling, Relative Quantification, and Identification of Sialylated N-Linked Oligosaccharides by UPLC-FLR-ESI/MS After Derivatization with Fluorescent Anthranilamide • Claire I. Butré, Eric Largy, Fabrice Cantais, and Arnaud Delobel
	1 Introduction
	2 Materials
		2.1 Relative Quantification of the Sialylation of N-Glycan by UPLC-FLR
			2.1.1 Apparatus
			2.1.2 UPLC Preparation
			2.1.3 Sample Preparation
		2.2 Charge-Based Profiling of N-Glycans by UPLC-FLR-MSE
			2.2.1 Apparatus
			2.2.2 UPLC Preparation
			2.2.3 Sample Preparation
	3 Methods
		3.1 Relative Quantification of the Sialylation of N-Glycan by UPLC-FLR
			3.1.1 Sample Preparation (One-Step Digestion)
			3.1.2 Sample Preparation (Two-Step Digestion)
			3.1.3 UHPLC Separation
			3.1.4 Data Analysis
		3.2 Charge-Based Profiling of N-Glycans by UPLC-FLR-MSE
			3.2.1 Sample Preparation
			3.2.2 UHPLC Separation
			3.2.3 MS Conditions
			3.2.4 Data Analysis
	4 Notes
	References
18. Linkage Analysis of Oligosaccharides and Polysaccharides: A Tutorial • Ian Black, Christian Heiss, Russell W. Carlson, and Parastoo Azadi
	1 Introduction
		1.1 Chemistry of the Linkage Analysis
	2 Materials
		2.1 Preacetylation
		2.2 Methylation
		2.3 Reduction of Uronic Acid Methyl Ethers
		2.4 Hydrolysis
		2.5 Reduction
		2.6 Acetylation
		2.7 GC-MS Analysis
	3 Methods
		3.1 Preacetylation
		3.2 Methylation
			3.2.1 NaOH Base Preparation
			3.2.2 Potassium Dimsyl Base Preparation
			3.2.3 Methylation of the Sample with NaOH  Base
			3.2.4 Methylation of the Sample with Dimsyl  Base
		3.3 Reduction of Uronic Acid Methyl Ethers
		3.4 Hydrolysis
		3.5 Reduction
		3.6 Acetylation
			3.6.1 For Neutral Partially Methylated Alditols
			3.6.2 For Amino Containing Sugars
		3.7 GC-MS Analysis
		3.8 Interpretation of Spectra
		3.9 Example Chromatograms
		3.10 Conclusion
	4 Notes
	References
19. Use of Exoglycosidases for the Structural Characterization of Glycans • Elizabeth McLeod, Paula Magnelli, and Xiaofeng Shi
	1 Introduction
	2 Materials
	3 Methods
		3.1 Protocol Using Procainamide (PCA) or 2-Aminobenzamide (2AB)
			3.1.1 Rapid Deglycosylation
			3.1.2 Fluorescent Labeling with Procainamide (PCA) or 2-Aminobenzamide (2AB)
			3.1.3 Glycan Purification with a 96-Well HILIC Plate
			3.1.4 Glycan Purification with a HILIC Spin Column
			3.1.5 Digestion of PCA or 2AB Labeled Glycans with Exoglycosidases
		3.2 Protocol Using Waters GlycoWorks RapiFluor-MS N-Glycan Kit
			3.2.1 Rapid Deglycosylation
			3.2.2 Fluorescent Labeling of Released Glycans with RapiFluor
			3.2.3 Glycan Purification with a 96-Well HILIC Plate
			3.2.4 Digestion of RapiFluor-Labeled Glycans with Exoglycosidases
	4 Notes
	Reference
20. Determination of Isomeric Glycan Structures by Permethylation and Liquid Chromatography-Mass Spectrometry (LC-MS) • Byeong Gwan Cho, Alireza Banazadeh, Wenjing Peng, Jingfu Zhao, Mona Goli, Sakshi Gautam, Ahmed Hussein, and Yehia Mechref
	1 Introduction
	2 Materials
		2.1 Protein Extraction
		2.2 N-Glycan Release
			2.2.1 In-Solution N-Glycan Release
			2.2.2 Filter Aided N-Glycan Release
		2.3 O-Glycan Release
		2.4 Glycan Purification
			2.4.1 Sodium Deoxycholate Removal
			2.4.2 Protein Precipitation
			2.4.3 Dialysis
			2.4.4 C18 Glycan Purification
		2.5 Reduction of Reducing End of Glycans
		2.6 Solid-Phase Permethylation
		2.7 PGC-LC Separation
		2.8 Mass Spectrometry
	3 Methods
		3.1 Protein Extraction
			3.1.1 Cell Line Protein Extraction
		3.2 N-Glycan Release
			3.2.1 In-Solution N-Glycan Release
			3.2.2 Filter-Aided N-Glycan Release
		3.3 O-Glycan Release
			3.3.1 Pronase Digestion
			3.3.2 Reductive β-Elimination
			3.3.3 Solid-Phase Permethylation
		3.4 Glycan Purification
			3.4.1 Sodium Deoxycholate Removal (For Cell Line and Tissue Samples)
			3.4.2 Protein Precipitation
			3.4.3 Dialysis (See Note 4)
			3.4.4 C18 Glycan Purification (See Note 5)
		3.5 Reduction of the Reducing End of Glycans
		3.6 Solid-Phase Permethylation
		3.7 PGC-LC Conditions
		3.8 MS Conditions
		3.9 Data Processing
			3.9.1 Glycan Isomer Identification and Quantitation
		3.10 Glycan Isomer Identification
			3.10.1 Identification by Comparison to NMR  Data
			3.10.2 Identification Using Exoglycosidase Digestion
			3.10.3 Identification Using MSn  Data
			3.10.4 Identification Using Standard Glycan Isomers
			3.10.5 Identification Using Modeling  Data
	4 Notes
	References
21. Optimization of Multiple Glycosidase and Chemical Stabilization Strategies for N-Glycan Isomer Detection by Mass Spectrometry Imaging in Formalin-Fixed, Paraffin-Embedded Tissues • Connor A. West, Xiaowei Lu, Grace Grimsley, Kim Norris-Caneda, Anand S. Mehta, Peggi M. Angel, and Richard R. Drake
	1 Introduction
	2 Materials
		2.1 Solutions for MALDI Imaging Mass Spectrometry
		2.2 Tissue Clearing Solution
		2.3 Enzyme and TM-Sprayer Solutions
		2.4 Amidation Reaction Solutions
	3 Methods
		3.1 Heating and Dewaxing
		3.2 Slide Scanning
		3.3 Amidation-Amidation (AA) Reaction
		3.4 Antigen Retrieval
		3.5 Endo F3 Application by the TM-Sprayer
		3.6 Incubation for On-Tissue Digestion
		3.7 MALDI Matrix Application by the TM-Sprayer
		3.8 Tissue Clearing of Matrix and Residual Endo F3 Cleaved N-Glycans
	4 Notes
	References
22. Glycosylation Profiling of Glycoproteins Secreted from Cultured Cells Using Glycan Node Analysis and GC-MS • Jesús S. Aguilar Díaz de león and Chad R. Borges
	1 Introduction
	2 Materials
		2.1 Cell Culture and Antibody
		2.2 Concentration of Cell Culture Media
		2.3 Glycan Node Analysis
	3 Methods
		3.1 Cell Culture
		3.2 Concentration of Cell Media by Spin Filtration
		3.3 IgG (Alpha-1 Anti-Trypsin) Antibody Desialylation & Preparation
		3.4 Glycan Node Analysis
	4 Notes
	References
23. Array-Based N-Glycan Profiling of Cells in Culture • Peggi M. Angel, Anand S. Mehta, and Richard R. Drake
	1 Introduction
	2 Materials
		2.1 Sample Preparation Solutions
		2.2 Enzyme Solutions
		2.3 TM-Sprayer Solutions
	3 Methods
		3.1 Cell Preparation for MS Profiling
		3.2 Slide Scanning
		3.3 PNGase F Application by the M3 TM-Sprayer
		3.4 Incubation for On-Tissue Digestion
		3.5 MALDI Matrix Application by the M3 TM-Sprayer
		3.6 Array Application of Ammonium Phosphate Monobasic (AP) Solution
		3.7 Mass Spectrometry Profiling
		3.8 Post-Acquisition Cell Measurements
	4 Notes
	References
24. Analysis of Oligomeric and Glycosylated Proteins by Size-Exclusion Chromatography Coupled with Multiangle Light Scattering • Kathryn Hastie, Vamseedhar Rayaprolu, and Erica Ollmann Saphire
	1 Introduction
	2 Materials
		2.1 Instruments and LC Column (See Note 1)
		2.2 Reagents and Supplies
	3 Methods
		3.1 System Setup
		3.2 System and Column Equilibration
		3.3 System Configuration and Validation
		3.4 Determination of the Molar Mass of a Glycoprotein Using Protein Conjugate Analysis in ASTRA
		3.5 Determination of the Molar Mass of a Glycoprotein-Fab Complex Protein Conjugate Analysis in ASTRA
	4 Notes
	References
25. Analysis of Glycoproteins by ATR-FTIR Spectroscopy: Comparative Assessment • Allison Derenne, Kheiro-Mouna Derfoufi, Ben Cowper, Cédric Delporte, Claire I. Butré, and Erik Goormaghtigh
	Abbreviations
	1 Introduction
	2 Materials
		2.1 Chemicals
		2.2 Equipment
		2.3 Samples
	3 Methods
		3.1 Preparation of the FTIR Spectrometer and the  IRE
		3.2 Sample Spreading and Spectrum Recording
		3.3 Spectral Processing
		3.4 Spectral Analysis: Comparison of the Global Glycosylation Level
		3.5 Spectral Analysis: Comparison of the Glycan Composition
	4 Notes
	References
26. Deploying Mass Spectrometric Data Analysis in the Amazon AWS Cloud Computing Environment • Jonathan E. Katz
	1 Introduction
	2 Materials
	3 Methods
		3.1 Creation of an Amazon AWS Account
		3.2 Log into Your Account
		3.3 Optional Additional Account Configurations
		3.4 Create Your First Data Drive
		3.5 Creation of Computer Credentials
		3.6 ``Provision´´ Your First Compute Instance
		3.7 Connecting to Your New Cloud Computer
		3.8 Attach and Configure Your Data Drive
		3.9 Transferring Data to Your New Computer (Including Vendor Software)
		3.10 Creating Snapshots
		3.11 Workflow Examples
			3.11.1 Clone Your OS Drive and Create a New Compute Instance from  it
			3.11.2 Increase the Size of Your OS Volume
			3.11.3 Create a Data Analysis System for a Collaborator
			3.11.4 Cheap Laptops and Cloud Resources Make for Safer and More Robust Bench-Side Computing
	4 Notes
	References
Index