Mucins: Methods and Protocols (Methods in Molecular Biology, 2763)

Preface
Contents
Contributors
Part I: Extraction and Separation
	Chapter 1: Preparation of Jellyfish Mucin
		1 Introduction
		2 Materials
			2.1 Catching  JF
			2.2 Storage of Raw  JF
			2.3 Extraction of SP Q-mucin
			2.4 Anion Exchange Chromatography (AEXC)
			2.5 Batch Purification by Adsorption and Desorption on AEX Resin
			2.6 Amino Acids Composition Analysis (AACA)
			2.7 Monosaccharide Composition Analysis
			2.8 Glycan Chain (Oligosaccharides) Composition Analysis After Hydrazinolysis
			2.9 Glycan Composition Analysis by β-elimination
			2.10 Identification of Each Single TR with Limited Degradation
			2.11 Quantitation of Sulfate Group
			2.12 Molecular Mass Estimation
		3 Methods
			3.1 Catching and Storage of  JFs
			3.2 Extraction of SP Q-mucin
			3.3 Anion Exchange Chromatography (AEXC)
				3.3.1 AEXC with Continuous Ion Gradient Program (Including Fractionation)
				3.3.2 AEXC with Stepwise Ion Gradient Program (Including Fractionation)
				3.3.3 Batch Purification Based on the Adsorption and Desorption on the Anion Exchange Resin
			3.4 Amino Acid Composition Analysis (AACA)
			3.5 Glycan Chain Analysis
				3.5.1 Monosaccharide Analysis After Hydrolysis
				3.5.2 Glycan Chain Analysis After Hydrazinolysis
				3.5.3 Glycan Chain Analysis After β-elimination
				3.5.4 Summary of Glycan Analysis
			3.6 Limited Degradation of the Main Peptide Chain
			3.7 Quantitation of Sulfate Groups and Other Acidic Moieties
			3.8 Estimation of Molecular  Mass
		4 Notes
		References
	Chapter 2: Extraction of Mucin from Rodent Feces and Determination of O-Linked Oligosaccharide Chain Equivalent Derived from F...
		1 Introduction
		2 Materials
			2.1 Mucin Extraction
			2.2 Fecal Mucin Assay
		3 Methods
			3.1 Extraction of Mucins from Rodent Feces
			3.2 Determination of Fecal Mucin
				3.2.1 Dilution of the Sample Prepared from Feces
				3.2.2 Determination of O-Linked Oligosaccharide Chain Equivalent
			3.3 Calculation of Fecal Mucin Concentration Expressed as the Equivalent of O-Linked Oligosaccharide Chain
		4 Notes
		References
	Chapter 3: Preparation of Soluble Mucin Solutions from the Salivary Glands
		1 Introduction
		2 Materials
		3 Methods
		4 Notes
		References
	Chapter 4: Isolation of Membrane Bound Mucins from Human Bronchial Epithelial Cells
		1 Introduction
		2 Materials
			2.1 Collecting and Storing Washings
			2.2 Cesium Chloride Isopycnic Density Gradient Centrifugation
			2.3 Unloading the Gradient and Running a Slot/Dot Blot
		3 Methods
			3.1 Collecting HBE Cell Culture Washings
			3.2 Cesium Chloride Isopycnic Density Gradient Centrifugation
			3.3 Unloading the Gradient and Running a Slot/Dot Blot
		4 Notes
		References
	Chapter 5: Extraction and Fractionation of Human Gastric Mucins from Gastric Juice
		1 Introduction
		2 Materials
			2.1 Collection of Gastric Juice by Endoscopy
			2.2 Extraction of Human Gastric Mucins
			2.3 Fractionation of Human Gastric Mucins
		3 Methods
			3.1 Gastric Aspiration with the EGT (Endoscopic Gastrin Test) Technique
			3.2 Extraction and Measurement of Mucin in Gastric Juice
				3.2.1 Method I-Gel Filtration Method
				3.2.2 Method II-EtOH Precipitation Method (see Note 20)
			3.3 Fractionation of Human Gastric Mucin (see Note 26)
		4 Notes
		References
	Chapter 6: Extraction of Mucins from the Mammalian Intestinal Tract
		1 Introduction
		2 Materials
			2.1 Extraction of Mucins with Chaotropic Agents
			2.2 Protein Measurement and Identification
		3 Methods
			3.1 Extraction of Mucins with Chaotropic Agents from the Surface Epithelium
			3.2 Extraction of Mucins with Chaotropic Agents from the Whole Intestinal Tissue
			3.3 Protein Measurement and Identification
		4 Notes
		References
	Chapter 7: Supported Molecular Matrix Electrophoresis
		1 Introduction
		2 Materials
			2.1 Preparation of Mucin Samples for SMME
			2.2 SMME Membrane
			2.3 Membrane Electrophoresis
			2.4 Alcian Blue Staining
			2.5 Lectin Staining
			2.6 Glycan Analysis (see Note 21)
		3 Methods
			3.1 Preparation of Mucin Samples for SMME
				3.1.1 Porcine Stomach Mucin Sample as a Reference Material for SMME
				3.1.2 Intestinal Mucin Sample for SMME
			3.2 Preparation of the SMME Membrane
			3.3 Membrane Electrophoresis
			3.4 Alcian Blue Staining
			3.5 Lectin Staining
			3.6 Glycan Analysis
				3.6.1 Releasing O-glycan from SMME Bands
				3.6.2 Permethylation and MALDI MS
		4 Notes
		References
Part II: Staining, Detection, and Quantitation
	Chapter 8: Supported Immunohistochemical Staining of Mucins
		1 Introduction
		2 Materials
			2.1 Reagents for Immunohistochemistry
			2.2 Oher Solutions for Immunohistochemistry
			2.3 Instruments and Others
			2.4 Materials for In Situ Hybridization
		3 Methods
			3.1 Preparation of the Good Tissue Slides for IHC
			3.2 Deparaffinization, Rehydration and Blocking of Endogenous Peroxidase
			3.3 Primary Antibody Incubation
			3.4 Detection
			3.5 Mounting and Examination
			3.6 In Situ Hybridization
		4 Notes
		References
	Chapter 9: Succinylation-Alcian Blue Staining of Mucins on Polyvinylidene Difluoride Membrane
		1 Introduction
		2 Materials
			2.1 Pretreatment of Membrane (See Note 1)
			2.2 Succinylation-Alcian Blue Staining
			2.3 Image Capture
		3 Methods
			3.1 Pretreatment of Membrane
			3.2 Succinylation-Alcian Blue Staining
			3.3 Image Capture (Also See Chap. 7)
		4 Notes
		References
	Chapter 10: Quantitation of Mucin by Densitometry of an Alcian Blue-Stained Membrane
		1 Introduction
		2 Materials
		3 Methods
			3.1 Quantitation of Mucin
		4 Notes
		References
	Chapter 11: Quantitation of MUC5AC and MUC5B by Stable Isotope Labeling Mass Spectrometry
		1 Introduction
		2 Materials
			2.1 Sample Preparation
			2.2 LC-MS and Data Analysis
		3 Methods
			3.1 Sample Preparation and Storage
			3.2 Reduction, Alkylation, and Trypsin Digestion by  FASP
			3.3 Stable Isotope Labeling Mass Spectrometry
			3.4 Results Analysis
		4 Notes
		References
Part III: Preparation and Analysis of Mucin Glycans
	Chapter 12: Preparation of O-Glycans from Mucins Using Hydrazine Treatment
		1 Introduction
		2 Materials
			2.1 Mucins and Model Glycoproteins
			2.2 Hydrazine Treatment
			2.3 Analysis of O-Glycans
		3 Methods
			3.1 Release of O-glycan Using Hydrazine Treatment
			3.2 Analysis of O-Glycans
		4 Notes
		References
	Chapter 13: Eliminative Oximation of O-Glycans from Mucins
		1 Introduction
		2 Materials
			2.1 Eliminative Oximation
			2.2 Reductive Amination with 2-Aminobenzamide (2-AB)
		3 Methods
			3.1 Eliminative Oximation
			3.2 Reductive Amination with 2-Aminobenzamide (2-AB) (See Note 14)
		4 Notes
		References
	Chapter 14: 9-Fluorenylmethyl Chloroformate Labeling for O-Glycan Analysis
		1 Introduction
		2 Materials
			2.1 Recovery of Glycosylamine-Form O-Glycans from Mucin Using Ammonia-Based β-Elimination
			2.2 Fmoc Labeling of Glycosylamine-Form O-Glycans
			2.3 HILIC LC-FD Analysis of Fmoc-Labeled O-Glycans
			2.4 Recovery of Glycosylamine-Form and Free-Form O-glycans by Delabeling
			2.5 Fabrication of Glycan Array with Glycosylamine-Form O-Glycans
		3 Methods
			3.1 Preparation of Glycosylamine-Form O-Glycans by Ammonia-Based β-Elimination
			3.2 Fmoc Labeling of Glycosylamine-Form O-Glycans
			3.3 Analysis of Fmoc-Labeled O-Glycans by HILIC LC-FD
			3.4 Recovery of Glycosylamine-Form and Free-Form O-Glycans Using Fmoc Elimination
			3.5 Fabrication of O-Glycan Arrays Using Glycosylamine-Form O-Glycans
		4 Notes
		References
	Chapter 15: Liquid Chromatography and Capillary Electrophoresis Analysis of 2AA-Labeled O-Glycans
		1 Introduction
		2 Materials
			2.1 2AA Derivatization and Purification
			2.2 HILIC LC-FD Analysis
			2.3 Capillary Gel Electrophoresis
			2.4 Capillary Affinity Electrophoresis and Online Concentration for  CGE
		3 Methods
			3.1 2AA-Labeling and Purification (See Note 11)
			3.2 Analysis of 2AA-Labeled O-glycans Using HILIC LC-FD
			3.3 Analysis of 2AA-Labeled O-glycans Using  CGE
			3.4 Analysis of 2AA-Labeled O-glycans Using  CAE
			3.5 Online Concentration for  CGE
		4 Notes
		References
	Chapter 16: Preparation of Mucin Glycopeptides by Organic Synthesis
		1 Introduction
		2 Materials
			2.1 Glycopeptide Synthesis by SPPS (Solid-Phase Peptide Synthesis)
			2.2 Deprotection of Protecting Groups on Carbohydrate Components
			2.3 Purification and Analysis Components
		3 Methods
			3.1 Glycopeptide Synthesis by SPPS (Solid-Phase Peptide Synthesis)
			3.2 Deprotection of Protecting Groups on Carbohydrates
			3.3 Purification and Analysis
		4 Notes
		References
	Chapter 17: MALDI-TOF MS/MS Analysis of Permethylated O-Glycan Alditols Released from Mucins
		1 Introduction
		2 Materials
			2.1 Release of O-Glycans
			2.2 Permethylation of O-Glycan
			2.3 MALDI-TOF MS and MS/MS Analysis
		3 Methods
			3.1 Release of O-Glycans from Mucin by Alkaline Borohydride Treatment
			3.2 Preparation of Permethylation Reagent
			3.3 Permethylation of O-Glycans
			3.4 MALDI-TOF MS and MALDI-TOF MS/MS Analysis of Permethylated O-Glycans
			3.5 Mass Data Interpretation
		4 Notes
		References
	Chapter 18: Structural Elucidation of Sialylated O-Glycan Alditols Obtained from Mucins by Mass Spectrometry
		1 Introduction
		2 Materials
			2.1 Mucin Preparation
			2.2 Glycan Preparation
			2.3 Glycan Analysis
		3 Methods
			3.1 Preparation of Small Intestinal Mucins
			3.2 Purification of Glycans
				3.2.1 Preparation of O-Glycan Alditols
				3.2.2 Preparation of Acidic O-Glycan Alditols
				3.2.3 First-Step  HPLC
				3.2.4 Second-Step  HPLC
			3.3 Glycan Analysis
				3.3.1 MS/MS Analysis of Sialylated O-Glycan Alditols
				3.3.2 MS Analysis of Sialic Acid-Containing Branch
		4 Notes
		References
	Chapter 19: Differential Glycoform Analysis of MUC1 Derived from Biological Specimens Using an Antibody-Overlay Lectin Microar...
		1 Introduction
		2 Materials
			2.1 IHC of MUC1
			2.2 LCM
			2.3 Protein Extraction from Dissected Tissue Fragments
			2.4 IP of MUC1
			2.5 Antibody-Overlay  LMA
		3 Methods
			3.1 IHC Staining with Anti-MUC1 Antibody
			3.2 Tissue  LCM
			3.3 Protein Extraction
			3.4 MUC1  IP
			3.5 Antibody-Overlay  LMA
		4 Notes
		References
	Chapter 20: ISOGlyP: O-Glycosylation Site Prediction Using Peptide Sequences and GALNTs
		1 Introduction
		2 Methods
			2.1 Mucin-Type O-Glycosylation Prediction
			2.2 Selective Peptide Identification
		3 Notes
		References
Part IV: Molecular Biology
	Chapter 21: Assessment of Mucin-Associated Gene Expression Levels on the Ocular Surface
		1 Introduction
		2 Materials
			2.1 Impression Cytology
			2.2 Messenger RNA Extraction
			2.3 Real-Time RT-PCR
		3 Methods
			3.1 Impression Cytology
			3.2 Messenger RNA Extraction
			3.3 Real-Time RT-PCR
		4 Notes
		References
	Chapter 22: Methylation-Specific Electrophoresis
		1 Introduction
		2 Materials
			2.1 DNA Sample Preparation
			2.2 Electrophoresis
		3 Methods
			3.1 Sample Preparation
			3.2 Gel Preparation
			3.3 Electrophoresis
			3.4 Staining
		4 Notes
		References
	Chapter 23: Expression Analysis of Genes Corresponding to Mucins and Their Glycans from Cervical Tissue Using RNA Sequencing
		1 Introduction
		2 Materials
			2.1 Cervical Tissue Collection
			2.2 RNA Extraction from Cervical Tissue
			2.3 Library Preparation and RNA Sequencing
			2.4 Differential Expression Analysis
		3 Methods
			3.1 Sample Size Calculation and Tissue Collection from the Cervix
			3.2 Preparing an RNAse-Free Environment
			3.3 Tissue Processing and RNA Extraction
			3.4 Library Preparation and RNA Sequencing
			3.5 Differential Gene Expression Analysis
			3.6 Functional and Pathway Enrichment Analysis
		4 Notes
		References
	Chapter 24: Recombinant Production of Glycoengineered Mucins in HEK293-F Cells
		1 Introduction
			1.1 CRISPR/Cas9-Mediated Cellular Glycoengineering
			1.2 Generation of Mucin-Producing HEK293-F Cells Using The PiggyBac Transposon System
		2 Materials
			2.1 HEK293-F Cell Culture
			2.2 CRISPR/Cas9-Mediated Gene Editing
			2.3 Clonal Isolation, Selection, and Expansion
			2.4 Screening and Detection of Mutations
			2.5 Generation of Stable Cell Line for Recombinant Mucin Production
		3 Methods
			3.1 HEK293-F Cell Culture
				3.1.1 Thawing Cells
				3.1.2 Measurement of Cell Density and Viability
				3.1.3 Propagation of HEK293-F Suspension Cultures
				3.1.4 Generation of HEK293-F Conditioned Medium
				3.1.5 Cryopreservation
			3.2 CRISPR/Cas9-Mediated Gene Editing
				3.2.1 Design of CRISPR/Cas9 gRNA for Gene KO
				3.2.2 Manual Design of the ssDNA HDR Template
				3.2.3 Software-Assisted Design of the ssDNA HDR Template
				3.2.4 Transfection of HEK293-F Cells with CRISPR/Cas9 Machinery and HDR Template
				3.2.5 Clonal Expansion of HEK293-F Cells
			3.3 Screening for Homozygous KO Clones
				3.3.1 Polymerase Chain Reaction (PCR) Amplification of Genomic DNA
				3.3.2 Restriction Digestion of PCR Product
				3.3.3 Analysis of Digested Product on a DNA Gel
			3.4 Generation of Stable Cell Line for Recombinant Mucin Production in Glycoengineered or Parental HEK293-F Cells
				3.4.1 Stable Introduction of Mucin Expression Cassettes into HEK293-F Cells
				3.4.2 Isolation of High Mucin-Expressing Subpopulations by Fluorescence Activated Cell Sorting (FACS)
			3.5 Recombinant Mucin Production
		4 Notes
		References
Part V: Interaction of Mucins and Other Biomolecules
	Chapter 25: Analysis of the Interaction Between Mucin and Green Fluorescent Protein (GFP)-Tagged Galectin-2 Using a 96-Well Pl...
		1 Introduction
		2 Materials
			2.1 Reagents for Preparation of Recombinant Gal-2-GFP
			2.2 Analysis of the Interaction Between Mucin and Gal-2-GFP
		3 Methods
			3.1 Preparation of Recombinant Gal-2-GFP
			3.2 Standard Curve of Gal-2-GFP
			3.3 Analysis of the Interaction Between Mucin and Gal-2-GFP Using a 96-Well Plate
		4 Notes
		References
	Chapter 26: Solution NMR Analysis of O-Glycopeptide-Antibody Interaction
		1 Introduction
		2 Materials
		3 Methods
			3.1 Sample Preparation for NMR Study
			3.2 NMR Signal Assignment of O-Glycopeptide
			3.3 NMR Titration Study
		4 Notes
		References
Part VI: Mucin and Microorganism
	Chapter 27: Cultivation of Microorganisms in Media Supplemented with Mucin Glycoproteins
		1 Introduction
		2 Materials
		3 Methods
			3.1 Pre-culture of B. bifidum
			3.2 Preparation of the Mucin Medium and Cultivation
		4 Notes
		References
	Chapter 28: Bacterial Enzyme Assay for Mucin Glycan Degradation
		1 Introduction
		2 Materials
			2.1 Enzyme Reaction
			2.2 TLC Components
			2.3 Quantification of GlcNAc-6S by LC-MS/MS
		3 Methods
			3.1 Enzyme Reaction with Mucin
			3.2 Detection of Released N-Acetylneuraminic Acid by TLC
			3.3 Measurement of GlcNAc-6S by LC-MS/MS
				3.3.1 Preparation of the Samples
				3.3.2 LC-MS/MS Analysis
		4 Notes
		References
	Chapter 29: Measurement of Mucinase Activity
		1 Introduction
		2 Materials
			2.1 Fecal Pellet Preparation
			2.2 Fecal Mucinase Assay
			2.3 Reducing Sugar Measurement
			2.4 Nitrogen Determination
		3 Methods
			3.1 Fecal Pellet Preparation
			3.2 Fecal Mucinase Reaction
			3.3 Reducing Sugar Measurement
			3.4 Nitrogen Determination
				3.4.1 Kjeldahl Digestion
				3.4.2 Kjeldahl Distillation and Titration (Fig. 2)
			3.5 Calculation of Mucinase Activity
		4 Notes
		References
	Chapter 30: Adhesion Inhibition Assay for Helicobacter pylori to Mucin by Lactobacillus
		1 Introduction
		2 Materials
			2.1 Bacterial Culture
			2.2 Microtiter Plate and Mucin
			2.3 Bacterial Inhibition Assay
			2.4 Quantification of H. pylori
		3 Methods
			3.1 Bacterial Culture
			3.2 Immobilization of PGM on Microtiter Plate
			3.3 Bacterial Inhibition Assay
			3.4 Quantification of H. pylori by qPCR
		4 Notes
		References
Part VII: Imaging and MD Simulation of Mucins
	Chapter 31: Imaging of Mucin Networks with Atomic Force Microscopy
		1 Introduction
		2 Materials
			2.1 Preparing the Mucin Solution
				2.1.1 Purifying Mucin Networks with Cushion Ultracentrifugation
				2.1.2 Purifying Mucin Networks with Gel Permeation Chromatography (GPC)
			2.2 Depositing the Sample onto Mica
			2.3 Imaging the Sample with AFM/Image Optimization
		3 Methods
			3.1 Preparing the Mucin Solution
				3.1.1 Purifying Mucin Networks with Cushion Ultracentrifugation
				3.1.2 Purifying Mucin Networks with Gel Permeation Chromatography (GPC)
			3.2 Depositing the Mucin Solution onto Mica
			3.3 Imaging the Sample with the AFM/Image Optimization
		4 Notes
		References
	Chapter 32: Molecular Dynamics Simulation and Docking of MUC1 O-Glycopeptide
		1 Introduction
		2 Hardware and Software
			2.1 Computer for Running Discovery Studio
			2.2 Software
		3 Methods
			3.1 Molecular Dynamics Simulation of MUC1 O-Glycopeptide
			3.2 Homology Modeling of Fv Domain
			3.3 Docking Simulations of MUC1 Peptides-Antibody
		4 Notes
		References
Part VIII: Mucin-Hydrogel and Physicochemical Properties
	Chapter 33: Fabrication and Characterization of Mucin Nanoparticles for Drug Delivery Applications
		1 Introduction
		2 Materials
			2.1 Mucin Purification
			2.2 Methacrylic Anhydride Functionalization of Mucins
			2.3 Preparing DNA-Crosslinked Nanoparticles
			2.4 Preparing UV-Crosslinked Nanoparticles
			2.5 Dynamic Light Scattering and Electrophoretic Light Scattering
			2.6 Drug Release Tests
		3 Methods
			3.1 Porcine Gastric Mucin (PGM) Purification
			3.2 Functionalization of Mucins with Methacrylic Anhydride (muc-MA)
			3.3 Preparing DNA-Crosslinked Mucin Nanoparticles
			3.4 Preparing Covalently Crosslinked Mucin Nanoparticles
			3.5 NP Characterization by Dynamic Light Scattering and Electrophoretic Light Scattering
			3.6 Quantifying Drug Release from the  NPs
		4 Notes
		References
	Chapter 34: Evaluation of Rheological Properties of Saliva by Determining the Spinnbarkeit
		1 Introduction
		2 Materials
		3 Methods
			3.1 Unstimulated Whole Saliva Collection
			3.2 Stimulated Submandibular/Sublingual Gland Saliva Collection
			3.3 Protease Inhibitor Preparation and Addition in the Saliva Sample
			3.4 Setup of Neva Meter
			3.5 Sample Installation
			3.6 Measurement
		4 Notes
		References
	Chapter 35: Mechanical Characterization of Mucus on Intestinal Tissues by Atomic Force Microscopy
		1 Introduction
		2 Materials
			2.1 Mouse Colon Explant
			2.2 AFM Measurement and Analysis
		3 Methods
			3.1 Preparation of the Colon Explant
			3.2 AFM Calibration
			3.3 AFM Measurements of Biopsies
			3.4 AFM Data Analysis
		4 Notes
		References
Index