MODULE 4: Chapter 5.2 Flashcards
What role does genomics play in protein characterization?
Genomics helps characterize the structure and function of individual proteins through the expression of cloned gene sequences.
What is the primary biochemical method for identifying an unknown purified protein?
Determining its amino acid sequence biochemically.
What technique is commonly used for amino acid sequence analysis in proteomic methods?
2-D PAGE followed by amino acid sequence analysis.
Who developed the Edman degradation method?
Pehr Edman, a Swedish biochemist.
What reagent was originally used in Sanger’s protein sequencing method?
1-fluoro-2,4-dinitrobenzene.
What is Edman degradation primarily used for?
To deduce the amino acid sequence of short polypeptides.
What is the advantage of Edman degradation over Sanger’s method?
It does not require the input of additional protein after each round of cleavage.
What is the maximum length of peptides that can be sequenced using Edman degradation?
Up to 50 amino acid residues.
What types of protease enzymes are used to cleave proteins for Edman degradation?
- Trypsin
- Chymotrypsin
What does trypsin cleave on?
The carboxyl side of lysine and arginine residues.
What is tandem mass spectrometry used for?
To determine the amino acid composition of peptide fragments.
What are the two ionization techniques mentioned for mass spectrometry?
- Electrospray ionization (ESI)
- Matrix-assisted laser desorption/ionization (MALDI)
What is the basic principle behind mass spectrometry?
It measures the mass-to-charge ratio (m/z) of molecules.
What is solid-phase peptide synthesis used for?
To synthesize peptide antigens for antibody production and peptide-based therapeutic drugs.
Who developed the strategy for solid-phase peptide synthesis?
Bruce Merrifield.
What is the direction of synthesis in solid-phase peptide synthesis?
From the C terminus to the N terminus.
What is the first step in solid-phase peptide synthesis?
Attaching the C-terminal amino acid to a resin molecule.
What is Fmoc in the context of solid-phase peptide synthesis?
A blocking group (9-fluorenylmethoxycarbonyl) used on the N-terminal side of incoming amino acids.
What happens to the protecting groups in solid-phase peptide synthesis?
They are removed after the peptide synthesis is complete.
What is the typical length of peptides generated through solid-phase peptide synthesis?
15 to 25 amino acid residues.
True or False: Edman degradation can efficiently sequence long polypeptides without cleavage.
False.
Fill in the blank: The Edman degradation method provides protein sequence information based on chemical determinations of _______.
[N-terminal amino acids]
What is the significance of the 1958 Nobel Prize awarded to Frederick Sanger?
It was for determining the amino acid sequence of insulin.
What does the process of mass spectrometry involve measuring?
The mass-to-charge ratio (m/z) of molecules.
What are tryptic fragments?
Peptide fragments produced by trypsin cleavage.
What is Edman degradation?
A protein sequencing method based on labeling and cleaving the N-terminal residue without disrupting the rest of the polypeptide chain.
Edman degradation allows for the sequencing of proteins from the amino-terminal end.
What is the main advantage of mass spectrometry over Edman degradation for protein identification?
Complex mixtures of protein subfragments can be analyzed without prior purification.
This significantly reduces sample preparation time compared to Edman degradation.
What is a disadvantage of Edman degradation?
It only sequences proteins from the amino-terminal end and covers a small region of the total protein sequence.
This limits its overall effectiveness in determining the full sequence of large proteins.
What does mass spectrometry use to identify proteins?
Mass analysis of peptide fragments and computer algorithms to calculate probabilities.
This method relies on comparing unknown proteins to predicted protein sequences from a genomic database.
What is trypsin?
A protease that cleaves a polypeptide chain on the carboxyl side of lysine or arginine residues.
Trypsin is commonly used in protein digestion protocols.
What is chymotrypsin?
A protease that cleaves a polypeptide chain on the carboxyl side of tyrosine, tryptophan, and phenylalanine residues.
Chymotrypsin helps provide shorter polypeptide chains suitable for Edman degradation.
What does electrospray ionization (ESI) do?
Prepares proteins for mass spectrometry by providing highly charged peptide ions in the gas phase.
ESI is a key step in the mass spectrometry process.
What is matrix-assisted laser desorption/ionization (MALDI)?
A method for generating peptide ions for mass spectrometry that uses a laser to ionize the peptide fragments.
MALDI is an alternative ionization method used in mass spectrometry.
What is solid-phase peptide synthesis?
A technique to synthesize peptide antigens for antibody production and to manufacture peptide-based therapeutic drugs.
This method is widely used in the production of therapeutic peptides.
What would happen if the Fmoc protecting group was not added to amino acid 2 during peptide synthesis?
The product(s) after the first round of coupling would include unprotected amino acid 2.
This could lead to unwanted side reactions during subsequent coupling steps.
True or False: Mass spectrometry requires access to a genomic sequence database for protein identification.
True.
Without a genomic database, mass spectrometry cannot effectively identify proteins.
