Proteoform Identification: Methods and Protocols (Methods in Molecular Biology, 2500)

Preface
Contents
Contributors
Chapter 1: Proteoforms and Proteoform Families: Past, Present, and Future
	1 Introduction
	2 Separations
	3 Bioinformatic Tools
	4 Applications
	References
Chapter 2: Membrane Ultrafiltration-Based Sample Preparation Method and Sheath-Flow CZE-MS/MS for Top-Down Proteomics
	1 Introduction
	2 Materials
		2.1 Prepare Protein for MU Cleanup
		2.2 Membrane Ultrafiltration
		2.3 Prepare LPA (Linear Polyacrylamide)-Coated Capillary
		2.4 CZE-MS
	3 Method
		3.1 Prepare Protein for MU Cleanup
		3.2 Ultrafiltration Buffer Exchange
		3.3 Prepare Capillary for CE-MS
		3.4 CE-MS
	4 Notes
	References
Chapter 3: Size Exclusion Chromatography Strategies and MASH Explorer for Large Proteoform Characterization
	1 Introduction
	2 Materials
		2.1 Size Exclusion Chromatography
		2.2 Online Reverse-Phase Liquid Chromatography Mass Spectrometry
		2.3 MASH Explorer Data Analysis
	3 Methods
		3.1 Size Exclusion Chromatography
		3.2 Online Reverse-Phase Chromatography and Top-Down MS Analysis
		3.3 Data Analysis Using MASH Explorer´s Targeted  Mode
	4 Notes
	References
Chapter 4: RPLC-RPLC-MS/MS for Proteoform Identification
	1 Introduction
	2 Materials
		2.1 High-pH Mobile Phases (See Notes 1 and 2)
		2.2 Low-pH Mobile Phases (See Notes 1 and 2)
		2.3 HeLa Cell Culture and Lysis Reagents (See Note 4)
		2.4 E. coli Cell Culture and Lysis Reagents (See Note 5)
	3 Methods
		3.1 HeLa Cell Culture and Lysis (See Note 4)
		3.2 E. coli Cell Culture and Lysis
		3.3 Offline First-Dimension High-pH RPLC Separation and Fractionation
		3.4 Offline Second-Dimension Low-pH RPLC Separation and Top-Down MS/MS Analysis
		3.5 Online High-pH/Low-pH RPLC Separation and Top-Down MS/MS Analysis
		3.6 Protein and Proteoform Identification
	4 Notes
	References
Chapter 5: Monolithic Materials-Based RPLC-MS for Proteoform Separation and Identification
	1 Introduction
	2 Materials
		2.1 Fused Silica Capillary Pretreatment
		2.2 Monolithic Column Preparation
		2.3 Monolithic Column Functionalization
		2.4 Proteoform Separation and Identification
	3 Methods
		3.1 Pretreatment of the Fused Silica Capillary
		3.2 Synthesis of Ethylene-Bridged Hybrid Monolith in the Capillary
		3.3 ``Thiol-ene´´ Click Modification of Ethylene-Bridged Hybrid Monolith
		3.4 Functionalized Ethylene-Bridged Hybrid Monolithic Column Coupling to MS for ``Top-Down´´ Proteomics
		3.5 Data Analysis
	4 Notes
	References
Chapter 6: Capillary Isoelectric Focusing: Mass Spectrometry Method for the Separation and Online Characterization of Monoclon...
	1 Introduction
	2 Materials
	3 Methods
		3.1 Online CIEF-MS
		3.2 Protein Sample Treatment
		3.3 Protein Desalting and Buffer Exchange Procedure
		3.4 Capillary Cleaning Procedure
		3.5 Example Results
			3.5.1 Linearity of Established pH Gradient
			3.5.2 CIEF-MS Intact Charge Variant Characterization of mAbs
			3.5.3 Middle-Up CIEF-MS Charge Variant Characterization of mAb Subunits
	4 Notes
	References
Chapter 7: Proteoform Analysis and Construction of Proteoform Families in Proteoform Suite
	1 Introduction
	2 Data Preprocessing
		2.1 Deconvolution Results
		2.2 Top-Down Results
		2.3 Database
		2.4 Bottom-Up Results
		2.5 Mass and Retention Time Calibration
	3 Data Analysis
	4 Notes
	References
Chapter 8: Top-Down Mass Spectrometry Data Analysis Using TopPIC Suite
	1 Introduction
	2 Material
		2.1 Installation of ProteoWizard
		2.2 Installation of TopPIC Suite
		2.3 Preparation of Data Files
	3 Methods
		3.1 Convert Raw MS Files to mzML Files Using msconvert
		3.2 Spectral Deconvolution Using TopFD
		3.3 Proteoform Identification Using TopPIC
		3.4 Proteoform Identification Using TopMG
		3.5 Proteoform Abundance Comparison Using TopDiff
		3.6 Result Visualization Using TopMSV
	4 Troubleshooting
	5 Notes
	References
Chapter 9: Accurate Proteoform Identification and Quantitation Using pTop 2.0
	1 Introduction
	2 Materials
		2.1 General Workflow of pTop 2.0
		2.2 Step-by-Step Running Operations of pTop 2.0
			2.2.1 Installation and Activation of pTop 2.0
			2.2.2 The Main Interface of pTop 2.0
			2.2.3 Configuration of New Modifications
			2.2.4 Parameters in Identification
			2.2.5 Searching Results of pTop 2.0
	3 Methods
		3.1 Method of Tag-Based Identification of Truncated Proteins
		3.2 Identification and Characterization of Proteoforms
		3.3 Re-ranking and Filtering
		3.4 Protein Quantitation
	4 Results of pTop 2.0 on Three Test Data  Sets
		4.1 Data  Sets
		4.2 Proteoform Identifications on the Human Histone Data  Set
		4.3 Proteoform Identifications on the E. coli Data  Set
		4.4 Quantitative Analysis on the Yeast Data  Set
		4.5 Validation of FDR Estimates
		4.6 Analysis of the Running  Time
	5 Conclusion and Discussions
	References
Chapter 10: Proteoform Identification and Quantification Using Intact Protein Database Search Engine ProteinGoggle
	1 Introduction
		1.1 New Features of ESI Mass Spectra of Intact Proteins
		1.2 The Deconvolution (Deisotoping) Approach
	2 The IMEF Search Algorithm and Workflow
		2.1 Isotopic Mass-to-Charge Ratio and Envelope Fingerprinting
		2.2 Comprehensive Coverage of Dissociation Methods and Pathways
		2.3 Overlapping Isotopic Envelopes
		2.4 Neutral Loss and Internal Fragment  Ions
		2.5 The Intact Protein Database Search Engine ProteinGoggle
	3 Application of ProteinGoggle in Proteoform Identification
	4 Application of ProteinGoggle in Proteoform Quantification
	5 Conclusions and Perspectives
	References
Chapter 11: Mass Deconvolution of Top-Down Mass Spectrometry Datasets by FLASHDeconv
	1 Introduction
	2 Materials
	3 Methods
		3.1 Installation
		3.2 Input mzML File Generation
		3.3 Performing Basic Mass Deconvolution
		3.4 Adjusting Basic Parameters
		3.5 Adjusting Advanced Parameters
		3.6 Running FLASHDeconv in Other Environments
	4 Notes
	References
Chapter 12: Deconvolving Native and Intact Protein Mass Spectra with UniDec
	1 Introduction
	2 How Does UniDec  Work?
	3 Downloading and Installing UniDec
	4 UniDec Data Processing and Deconvolution
		4.1 Opening Mass Spectra
		4.2 UniDec Presets
		4.3 Data Processing
		4.4 Deconvolution Parameters and Quick Controls
		4.5 Advanced Deconvolution Parameters
	5 Analyzing Deconvolution  Data
		5.1 Peak Selection and Plotting
		5.2 Mass and Oligomer Tools for Peak Assignment
		5.3 Macromolecular Mass Defect Analysis
	6 Exporting Plots and Mass Spectra
	7 Conclusion
	8 Notes
	References
Chapter 13: Discovery of Unknown Posttranslational Modifications by Top-Down Mass Spectrometry
	1 Introduction
	2 Materials
		2.1 Online Reverse-Phase Liquid Chromatography (RPLC) Coupled to Mass Spectrometry
		2.2 Mass Spectrometry
		2.3 Data Analysis Software
	3 Methods
		3.1 Online RPLC Coupled with Mass Spectrometry Detection
		3.2 LC-MS Data Processing
		3.3 Identification and Confirmation of Unknown Proteoforms
	4 Notes
	References
Chapter 14: Determining Copper and Zinc Content in Superoxide Dismutase Using Electron Capture Dissociation Under Native Spray...
	1 Introduction
	2 Materials
		2.1 Reagents and Supplies
		2.2 Nanoelectrospray Ionization
		2.3 Mass Spectrometer and ExD  Cell
	3 Methods
		3.1 Protein Preparation
		3.2 Nanospray Ionization Conditions
		3.3 Mass Spectrometry
		3.4 ECD Fragment Ion Analysis
	4 Notes
	References
Chapter 15: Surface-Induced Dissociation for Protein Complex Characterization
	1 Introduction
	2 Equipment and Materials
		2.1 Self-Assembled Monolayer (SAM) Surface Preparation
		2.2 Offline Sample Preparation
		2.3 Pulling and Loading nESI Tips
		2.4 Calibration of Q Exactive UHMR
		2.5 SID on a Q Exactive UHMR
	3 Methods
		3.1 How Is SID Implemented in Different Mass Spectrometers?
		3.2 Incorporation of SID into a Thermo Scientific Q Exactive UHMR
		3.3 Preparing a Self-Assembled Monolayer Surface
		3.4 Sample Preparation for Native MS
			3.4.1 Buffer Exchange Spin Columns
			3.4.2 Concentration and Diafiltration
			3.4.3 Dialysis
			3.4.4 Online Buffer Exchange
		3.5 Pulling Nanospray Needles
		3.6 Getting Spray and Tuning the MS
			3.6.1 Mass Calibration
			3.6.2 Acquiring MS Spectra for Protein Complexes
			3.6.3 CID for Characterization of Protein Complex
		3.7 Tuning SID Over a Range of Energies
		3.8 Interpreting the Data
	4 Notes
	References
Index