Integrated methods in protein biochemistry. Part C /

Integrated Methods in Protein Biochemistry: Part B, Volume 678 in the Methods in Enzymology series, highlights new advances in the field, with this new volume presenting interesting chapters on a variety of topics, including Precise modification of native proteins, purification, and analysis of bioc...

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Detalles Bibliográficos
Clasificación:Libro Electrónico
Otros Autores: Shukla, Arun K. (Editor )
Formato: eBook
Idioma:Inglés
Publicado: Cambridge, MA : Academic Press, 2023.
Colección:Methods in enzymology ; v. 682.
Temas:
Proteins > Analysis > Research > Methodology.
Proteomics > Methodology.
Biochemistry.
Prot�eines > Analyse > Recherche > M�ethodologie.
Prot�eomique > M�ethodologie.
Biochimie.
biochemistry.
Biochemistry
Acceso en línea:Texto completo
Tabla de Contenidos:
  • Funding information
  • Conflict of interests
  • References
  • Chapter Three: Laminar flow-based microfluidic systems for molecular interaction analysis-Part 1: Chip development, syste ...
  • 1. Introduction
  • 2. Microfluidic device development
  • 2.1. Device material and fabrication
  • 2.2. Custom PDMS device design
  • 2.3. Channel aspect ratio
  • 2.4. Limitations of custom PDMS devices
  • 2.5. Post-fabrication treatment to prevent adsorption
  • 3. Fluidic actuation
  • 3.1. Choosing a suitable flow rate
  • 3.2. Controlling and measuring the flow rate
  • 3.3. On-chip fluid reservoirs
  • 4. Fluorescence detection
  • 4.1. Fluorescent labelling of proteins for analysis in the LaMInA system
  • 4.1.1. Fluorescein isothiocyanate (FITC)
  • 4.1.2. FITC labelling of DHDPS
  • 4.1.3. FITC labelling of rubisco
  • 4.1.4. FITC-labelling of protein size standards
  • 4.2. Alternative fluorescent dyes
  • 4.2.1. Ortho-phthalaldehyde
  • 4.2.2. Fluorescamine
  • 4.3. Technical considerations for the fluorescence detection hardware
  • 4.3.1. Colour charged-coupled device (CCD) camera
  • 4.3.2. Monochromatic scientific complementary metal oxide semiconductor (CMOS) camera
  • 4.3.3. Photomultiplier tube with CLS microscopy
  • 5. Summary and conclusions
  • References
  • Chapter Four: Spectroscopic analysis of the mammalian enzyme cysteine dioxygenase
  • 1. Introduction
  • 2. Spectroscopic analysis of CDO
  • 2.1. Resting state ferrous enzyme
  • 2.2. Substrate (analogue) bound CDO
  • 2.3. Substrate (analogue) bound CDO incubated with superoxide surrogates
  • 2.4. Secondary sphere CDO variants
  • Acknowledgments
  • References
  • Chapter Five: DeGlyPHER: Highly sensitive site-specific analysis of N-linked glycans on proteins
  • 1. Introduction
  • 1.1. N-glycosylated viral spike proteins as vaccine candidates
  • 1.2. Site-specific N-glycosylation analysis using HIV Env.
  • 1.3. Breaking the sensitivity barrier using a ``single-pot�� proteomic analysis
  • 1.3.1. Proteinase K
  • 1.3.2. Optimizing the reaction
  • 1.4. Sequential deglycosylation
  • 2. Before you begin
  • 2.1. Buffer exchange and concentrating the sample
  • 2.1.1. Buffer exchange
  • 2.1.2. Freezing
  • 2.1.3. Freeze-drying
  • 2.2. Preparing reaction solutions
  • 2.2.1. Solution X (for Endo H and Proteinase K).
  • 2.2.2. Solution Y (for PNGase F)
  • 2.3. Preparing proteinase K stock solution
  • 3. Key resources table
  • 4. Materials and equipment
  • 5. Step-by-step method details
  • 5.1. Reducing disulfide bridge(s)
  • 5.2. Endo H treatment at protein level
  • 5.3. Proteinase K digestion
  • 5.4. Endo H treatment at peptide level
  • 5.5. Freeze-drying the reaction and PNGase F treatment
  • 5.6. LC-MS/MS
  • 5.6.1. Q exactive HF-X with EASY-nLC 1200
  • 5.6.2. timsTOF Pro with Evosep One
  • 5.7. Data processing
  • 6. Quantification and statistical analysis
  • 6.1. Label-free quantitation
  • 6.2. Data analysis using GlycoMSQuant
  • 7. Expected outcomes
  • 8. Advantages
  • 9. Limitations
  • 10. Optimization and troubleshooting
  • 10.1. Validating efficiency of glycosidases
  • 10.2. Comparing PK vs trypsin
  • 10.3. DeGlyPHER vs our previous approach
  • 10.4. Analyzing DeGlyPHER�s limit of sensitivity
  • 10.5. Troubleshooting
  • 11. Safety considerations and standards
  • 12. Alternative methods/procedures
  • Acknowledgments
  • References
  • Chapter Six: Sample preparation for structural mass spectrometry via polyacrylamide gel electrophoresis
  • 1. Introduction
  • 2. Sample preparation for top-down mass spectrometry by PEPPI-MS
  • 2.1. Equipment
  • 2.2. Reagents
  • 2.3. Procedure
  • 2.4. Notes
  • 3. Sample preparation for native mass spectrometry by PEPPI-MS
  • 3.1. Equipment
  • 3.2. Reagent
  • 3.3. Procedure
  • 3.4. Notes.
  • 4. Sample preparation for cross-linking mass spectrometry by PEPPI-AnExSP
  • 4.1. Equipment
  • 4.2. Reagents
  • 4.3. Procedure
  • 4.4. Notes
  • 5. Summary and conclusions
  • Acknowledgments
  • References
  • Chapter Seven: High-throughput screening of glycosynthases using azido sugars for oligosaccharides synthesis
  • 1. Introduction
  • 2. Screening using cyn regulon based azide detection method
  • 2.1. Summary
  • 2.2. Preparation of error-prone PCR library
  • 2.2.1. Materials and equipment
  • 2.2.2. Procedure
  • 2.2.3. Notes
  • 2.3. Procedure for medium-throughput screening of mutants
  • 2.3.1. Materials and equipment
  • 2.3.2. Procedure
  • 2.3.3. Notes
  • 3. Screening using click chemistry based azide detection method
  • 3.1. Summary
  • 3.2. Preparation of error prone PCR library
  • 3.2.1. Materials and equipment
  • 3.2.2. Procedure
  • 3.2.3. Notes
  • 3.3. Procedure for high-throughput screening of mutants
  • 3.3.1. Materials and equipment
  • 3.3.2. Procedure
  • 3.4. Notes
  • 4. Summary and conclusions
  • Acknowledgments
  • References
  • Chapter Eight: Rational design, production and in vitro analysis of photoxenoproteins
  • 1. Introduction
  • 2. Rational design of photoxenoproteins
  • 3. Synthesis of phenylalanine-4-azobenzene AzoF
  • 3.1. Equipment
  • 3.2. Reagents
  • 3.3. Procedure
  • 4. Heterologous gene expression and purification of photoxenoproteins
  • 5. Characterization of photoxenoproteins
  • 5.1. Photochemical characterization of ONBY-containing POIs
  • 5.2. Photophysical characterization of AzoF-containing POIs
  • 5.2.1. Equipment
  • 5.2.2. Reagents
  • 5.2.3. Procedure
  • 6. Evaluation of photocontrol
  • 6.1. Photocontrol under steady-state conditions
  • 6.2. Photocontrol during turnover
  • 6.3. Example 1: Photocontrol of tryptophan synthase with ONBY
  • 6.4. Example 2: Photocontrol of imidazole glycerol phosphate synthase with AzoF.
  • 7. Notes
  • Acknowledgments
  • References
  • Chapter Nine: Chemical and structural approaches to investigate PTEN function and regulation
  • 1. Introduction
  • 2. Biochemical assays to determine the conformational state of PTEN
  • 3. Structural basis for N-terminal function and regulation
  • 3.1. Production of semi-synthetic PTENs for protein X-ray crystallography
  • 3.2. X-ray crystal structure of PTEN revealing N-terminal segment of PTEN
  • 3.3. Production of semi-synthetic N-terminally labeled PTEN for macromolecular NMR
  • 4. Structural basis for regulation of PTEN by C-terminal phosphorylation
  • 4.1. Production of VSP for macromolecular NMR experiments
  • 4.1.1. Isotopically labeling VSP for NMR assignments
  • 4.1.2. Protein semi-synthesis to generate segmentally labeled phosphorylated VSP for macromolecular NMR
  • 4.2. Structural model for how the C-terminal tail engages VSP
  • 5. Summary and future directions
  • Acknowledgments
  • References
  • Chapter Ten: Analyzing protein posttranslational modifications using enzyme-catalyzed expressed protein ligation
  • 1. Introduction
  • 2. General methods and statistical analysis
  • 3. Purification of subtiligase
  • 3.1. Materials and reagents
  • 3.1.1. For expression and purification
  • 3.2. Equipment
  • 3.3. Procedure
  • 3.3.1. Transformation
  • 3.3.2. Expression and purification
  • 3.4. Notes
  • 4. Synthesis of peptide using solid phase peptide synthesis (SPPS)
  • 4.1. Equipment
  • 4.2. Reagents
  • 4.3. Procedure (8-10h)
  • 4.4. Notes
  • 5. Generation of POI C-terminal thioester
  • 5.1. Materials and reagents
  • 5.1.1. For expression and purification
  • 5.2. Equipment
  • 5.3. Procedure
  • 5.3.1. Expression and purification of POI-MxeIntein-CBD fusion protein
  • 5.3.2. Intein cleavage
  • 5.4. Notes
  • 6. Enzyme-catalyzed EPL, and affinity purification of protein ligation product using biotin tag.

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