Methods of Protein Analysis Flashcards
The covalent peptide bonds that link amino acid residues within a polypeptide chain can be cleaved by harsh chemical conditions to generate
Individual amino acid residues
Individual amino acid residues can be:
- ) Separated by?
- ) Chemically modified to enable?
- ) Identified and quantified by their?
1.) Ion exchange chromatography 2.) Spectroscopic Detection 3.) Elution profiles
Amino acid composition of a protein is now easily available from translation of the DNA sequence of the
Corresponding Gene
The 20 amino acids commonly found in proteins are joined together by
Peptide Bonds
The sequence of amino acids in a protein is called the
Primary Structure
In proteins, amino acids are joined covalently by peptide bonds, which are amide linkages between the
α-carboxyl group of one amino acid and the α-amino group of another
Are not broken by conditions that denature proteins, such as heating or high concentrations of urea
Peptide Bonds
How can you break a peptide bond?
- ) Nonenzymically by hydrolysis or by prolonged exposure to a strong acid or base at elevated temp.
- ) With an enzyme
By convention, the free amino end (N-terminal) of the peptide chain is written to the
Left
All amino acid sequences are read from the
N- terminus to C-terminus
Each component amino acid in a polypeptide is called a “residue” because it is the portion of the amino acid remaining after the atoms of water are lost in the formation of the
Peptide bond
When a polypeptide is named, all amino acid residues have their suffixes (-ine, -an, -ic, or -ate) changed to
-With the exception of the C-terminal residue
-yl
Isomerically, the peptide bond is typically a
-Due to steric constraints
Trans bond
A purified sample of the polypeptide to be analyzed is first hydrolyzed by strong acid at
110°C for 24 hours.
In this technique, a mixture of amino acids is applied to a column that contains a resin to which a negatively charged group is tightly attached.
cation-exchange Chromatography
In cation-exchange chromatography, each amino acid is sequentially released from the chromatography column by eluting with solutions of increasing
Ionic strength and pH
The separated amino acids contained in the eluate from the column are quantitated by heating them with
-a reagent that forms a purple compound with most amino acids, ammonia, and amines.
Ninhydrin
The amount of each amino acid is determined spectrophotometrically by measuring the amount of light absorbed by the
Ninhydrin derivative
Can provide sequence information for ~15-50 residues at the N-terminus of a protein
Successive cycles of Edman Degradation
Typically sufficient to unambiguously identify a protein by comparison to genomic sequence data.
A sequence of fifteen N-terminal residues
Furthermore, N-terminal sequencing can be performed with very small quantities of protein such as an isolated band or spot of protein separated from a complex mixture of proteins by
One- or two-dimensional gel electrophoresis
A powerful method for protein identification and is also useful for identifying post-translational modifications in peptides isolated from a larger protein
N-terminal sequencing
N-terminal sequencing is rarely used for sequencing an entire protein because it is more efficient to obtain the protein sequence from the
DNA sequence of the corresponding gene
Known as Edman reagent, is used to label the amino-terminal residue under mildly alkaline conditions
Phenylisothiocyanate
Slowly hydrolyzes one peptide bond at a time, allowing N-terminal sequencing
Edman Degradation
Cleave the polypeptide backbone at sites of specific amino acid residues.
-Used to cleave proteins into smaller peptides.
Proteases (peptidases)
Two of the most commonly used proteases are the digestive enzymes
Trypsin and Chymotrypsin
Where does Trypsin cleave?
On the C-terminal side of basic residues (Arg, Lys)
Where does Chymotrypsin cleave?
On the C-terminal side of aromatic residues (Trp, Tyr, Phe)
Cut at the ends of proteins, and are divided into aminopeptidases (N-terminal cut) and carboxypeptidases (C-terminal cut).
Exopeptidases
Cleave within a protein
Endopeptidases
The movement of charged particles in an electric field
Electrophoresis
Widely used in biochemistry and molecular biology laboratories to separate macromolecules such as peptides, proteins, DNA and RNA.
Electrophoresis
If a mixture of different species are applied at a single point (origin) and subjected to an electrical field, the species will migrate linearly according to their
Net charge
In Electrophoresis, what is the
- ) Negative electrode?
- ) Positive electrode?
1.) Cathode 2.) Anode
Following electrophoresis in one direction, it is repeated in a perpendicular direction by rotating the electrophoretogram 90˚ relative to the electrodes.
Two-dimensional Electrophoresis
If conditions for the second electrophoresis are the same as the first, the species will migrate identically and will simply spread out along a
Diagonal
However, if the conditions are altered, the species will migrate differently in the second dimension, spreading out in a
Two-dimensional pattern
Small molecules such as amino acids and peptides can be separated by electrophoresis on
Paper
This is useful for two-dimensional electrophoresis of complex mixtures of peptides obtained by proteolytic cleavage of large proteins
Paper Electrophoresis performed by altering pH to change charge
Two-dimensional electrophoresis of peptides played an important role in identifying the molecular basis of
Sickle Cell disease
The speed of migration of a peptide is determined by both size and charge in
Polyacrylamide gel electrophoresis
Added to the polyacrylamide gel to achieve separation of proteins on the basis of mass alone
-an ionic detergent that denatures proteins and binds along the unfolded peptide chain, imparting a negative charge that is proportional to their mass
Sodium dodecyl sulfate (SDS)
Separates peptides on the basis of mass alone
SDS page
Used to separate proteins based on their isoelectric point (PI)
Isoelectric focusing gel electrophoresis
The pH at which a peptide carries no net electric charge
Isoelectric point
A powerful method for identifying a missing protein in tissue from a patient.
Two-dimensional Electrophoresis with Isoelectric focusing followed by SDS PAGE
Proteins separated by polyacrylamide gel electrophoresis are transferred from the gel to a membrane. The membrane is first incubated with antibodies that bind to a specific protein and then with a fluorescent, radioactive, or chemi-luminescent secondary antibody that binds to the first antibody, enabling visualization
Immunoblotting (western blot)
A large variety of mass spectrometry applications have been developed to identify and quantify proteins and to elucidate their chemical composition, including
Amino acid sequence and identification of post-translational modifications
The two methods available for determining three-dimensional structures of macromolecules at atomic resolution.
X-ray crystallography and NMR spectroscopy
NMR is typically only used for the determination of
Small proteins (
Which amino acid ridgifies the protein backbone?
Proline (P)
What are four amino acids that are charged at neutral pH?
Arginine, Aspartic Acid, Glutamic Acid, Lysine
How much would you expect a 90 residue protein to weigh?
10,000 Daltons
Distances are commonly measured in angstroms (1 x 10^-10 meters). An average sized protein (150 residues) will typically be between
30 and 50 angstroms wide
The average weight of an amino acid is
110 Daltons
Can form disulfide bonds between their two sulfur atoms
Cysteine pairs
Can be used to stabilize or constrain proteins and are typically found in extracellular proteins
Disulfide bonds
Have no side chains and as a result, are more flexible than other amino acids
-often found in tight turns within a protein
Glycine
Often used to make a peptide more rigid
Proline
