Methods of Protein Analysis

Question 1

The covalent peptide bonds that link amino acid residues within a polypeptide chain can be cleaved by harsh chemical conditions to generate

Answer 1

Individual amino acid residues

Question 2

Individual amino acid residues can be:

1. ) Separated by?
2. ) Chemically modified to enable?
3. ) Identified and quantified by their?

Answer 2

1.) Ion exchange chromatography 2.) Spectroscopic Detection 3.) Elution profiles

Question 3

Amino acid composition of a protein is now easily available from translation of the DNA sequence of the

Answer 3

Corresponding Gene

Question 4

The 20 amino acids commonly found in proteins are joined together by

Answer 4

Peptide Bonds

Question 5

The sequence of amino acids in a protein is called the

Answer 5

Primary Structure

Question 6

In proteins, amino acids are joined covalently by peptide bonds, which are amide linkages between the

Answer 6

α-carboxyl group of one amino acid and the α-amino group of another

Question 7

Are not broken by conditions that denature proteins, such as heating or high concentrations of urea

Answer 7

Peptide Bonds

Question 8

How can you break a peptide bond?

Answer 8

1. ) Nonenzymically by hydrolysis or by prolonged exposure to a strong acid or base at elevated temp.
2. ) With an enzyme

Question 9

By convention, the free amino end (N-terminal) of the peptide chain is written to the

Answer 9

Left

Question 10

All amino acid sequences are read from the

Answer 10

N- terminus to C-terminus

Question 11

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

Answer 11

Peptide bond

Question 12

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

Answer 12

-yl

Question 13

Isomerically, the peptide bond is typically a

-Due to steric constraints

Answer 13

Trans bond

Question 14

A purified sample of the polypeptide to be analyzed is first hydrolyzed by strong acid at

Answer 14

110°C for 24 hours.

Question 15

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.

Answer 15

cation-exchange Chromatography

Question 16

In cation-exchange chromatography, each amino acid is sequentially released from the chromatography column by eluting with solutions of increasing

Answer 16

Ionic strength and pH

Question 17

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.

Answer 17

Ninhydrin

Question 18

The amount of each amino acid is determined spectrophotometrically by measuring the amount of light absorbed by the

Answer 18

Ninhydrin derivative

Question 19

Can provide sequence information for ~15-50 residues at the N-terminus of a protein

Answer 19

Successive cycles of Edman Degradation

Question 20

Typically sufficient to unambiguously identify a protein by comparison to genomic sequence data.

Answer 20

A sequence of fifteen N-terminal residues

Question 21

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

Answer 21

One- or two-dimensional gel electrophoresis

Question 22

A powerful method for protein identification and is also useful for identifying post-translational modifications in peptides isolated from a larger protein

Answer 22

N-terminal sequencing

Question 23

N-terminal sequencing is rarely used for sequencing an entire protein because it is more efficient to obtain the protein sequence from the

Answer 23

DNA sequence of the corresponding gene

Question 24

Known as Edman reagent, is used to label the amino-terminal residue under mildly alkaline conditions

Answer 24

Phenylisothiocyanate

Question 25

Slowly hydrolyzes one peptide bond at a time, allowing N-terminal sequencing

Answer 25

Edman Degradation

Question 26

Cleave the polypeptide backbone at sites of specific amino acid residues. -Used to cleave proteins into smaller peptides.

Answer 26

Proteases (peptidases)

Question 27

Two of the most commonly used proteases are the digestive enzymes

Answer 27

Trypsin and Chymotrypsin

Question 28

Where does Trypsin cleave?

Answer 28

On the C-terminal side of basic residues (Arg, Lys)

Question 29

Where does Chymotrypsin cleave?

Answer 29

On the C-terminal side of aromatic residues (Trp, Tyr, Phe)

Question 30

Cut at the ends of proteins, and are divided into aminopeptidases (N-terminal cut) and carboxypeptidases (C-terminal cut).

Answer 30

Exopeptidases

Question 31

Cleave within a protein

Answer 31

Endopeptidases

Question 32

The movement of charged particles in an electric field

Answer 32

Electrophoresis

Question 33

Widely used in biochemistry and molecular biology laboratories to separate macromolecules such as peptides, proteins, DNA and RNA.

Answer 33

Electrophoresis

Question 34

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

Answer 34

Net charge

Question 35

In Electrophoresis, what is the 1. ) Negative electrode? 2. ) Positive electrode?

Answer 35

1.) Cathode 2.) Anode

Question 36

Following electrophoresis in one direction, it is repeated in a perpendicular direction by rotating the electrophoretogram 90˚ relative to the electrodes.

Answer 36

Two-dimensional Electrophoresis

Question 37

If conditions for the second electrophoresis are the same as the first, the species will migrate identically and will simply spread out along a

Answer 37

Diagonal

Question 38

However, if the conditions are altered, the species will migrate differently in the second dimension, spreading out in a

Answer 38

Two-dimensional pattern

Question 39

Small molecules such as amino acids and peptides can be separated by electrophoresis on

Answer 39

Paper

Question 40

This is useful for two-dimensional electrophoresis of complex mixtures of peptides obtained by proteolytic cleavage of large proteins

Answer 40

Paper Electrophoresis performed by altering pH to change charge

Question 41

Two-dimensional electrophoresis of peptides played an important role in identifying the molecular basis of

Answer 41

Sickle Cell disease

Question 42

The speed of migration of a peptide is determined by both size and charge in

Answer 42

Polyacrylamide gel electrophoresis

Question 43

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

Answer 43

Sodium dodecyl sulfate (SDS)

Question 44

Separates peptides on the basis of mass alone

Answer 44

SDS page

Question 45

Used to separate proteins based on their isoelectric point (PI)

Answer 45

Isoelectric focusing gel electrophoresis

Question 46

The pH at which a peptide carries no net electric charge

Answer 46

Isoelectric point

Question 47

A powerful method for identifying a missing protein in tissue from a patient.

Answer 47

Two-dimensional Electrophoresis with Isoelectric focusing followed by SDS PAGE

Question 48

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

Answer 48

Immunoblotting (western blot)

Question 49

A large variety of mass spectrometry applications have been developed to identify and quantify proteins and to elucidate their chemical composition, including

Answer 49

Amino acid sequence and identification of post-translational modifications

Question 50

The two methods available for determining three-dimensional structures of macromolecules at atomic resolution.

Answer 50

X-ray crystallography and NMR spectroscopy

Question 51

NMR is typically only used for the determination of

Answer 51

Small proteins (

Question 52

Which amino acid ridgifies the protein backbone?

Answer 52

Proline (P)

Question 53

What are four amino acids that are charged at neutral pH?

Answer 53

Arginine, Aspartic Acid, Glutamic Acid, Lysine

Question 54

How much would you expect a 90 residue protein to weigh?

Answer 54

10,000 Daltons

Question 55

Distances are commonly measured in angstroms (1 x 10^-10 meters). An average sized protein (150 residues) will typically be between

Answer 55

30 and 50 angstroms wide

Question 56

The average weight of an amino acid is

Answer 56

110 Daltons

Question 57

Can form disulfide bonds between their two sulfur atoms

Answer 57

Cysteine pairs

Question 58

Can be used to stabilize or constrain proteins and are typically found in extracellular proteins

Answer 58

Disulfide bonds

Question 59

Have no side chains and as a result, are more flexible than other amino acids -often found in tight turns within a protein

Answer 59

Glycine

Question 60

Often used to make a peptide more rigid

Answer 60

Proline