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Flashcards in Methods of Protein Analysis Deck (60):
1

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

2

Individual amino acid residues can be:

1.) Separated by?

2.) Chemically modified to enable?

3.) Identified and quantified by their?

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

3

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

Corresponding Gene

4

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

Peptide Bonds

5

The sequence of amino acids in a protein is called the

Primary Structure

6

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

7

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

Peptide Bonds

8

How can you break a peptide bond?

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

2.) With an enzyme

9

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

Left

10

All amino acid sequences are read from the

N- terminus to C-terminus

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

Peptide bond

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

-yl

13

Isomerically, the peptide bond is typically a

-Due to steric constraints

Trans bond

14

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

110°C for 24 hours.

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.

cation-exchange Chromatography

16

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

Ionic strength and pH

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.

Ninhydrin

18

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

Ninhydrin derivative

19

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

Successive cycles of Edman Degradation

20

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

A sequence of fifteen N-terminal residues

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

One- or two-dimensional gel electrophoresis

22

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

23

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

24

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

Phenylisothiocyanate

25

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

Edman Degradation

26

Cleave the polypeptide backbone at sites of specific amino acid residues.

-Used to cleave proteins into smaller peptides.

Proteases (peptidases)

27

Two of the most commonly used proteases are the digestive enzymes

Trypsin and Chymotrypsin

28

Where does Trypsin cleave?

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

29

Where does Chymotrypsin cleave?

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

30

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

Exopeptidases

31

Cleave within a protein

Endopeptidases

32

The movement of charged particles in an electric field

Electrophoresis

33

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

Electrophoresis

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

Net charge

35

In Electrophoresis, what is the

1.) Negative electrode?

2.) Positive electrode?

1.) Cathode 2.) Anode

36

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

Two-dimensional Electrophoresis

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

Diagonal

38

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

Two-dimensional pattern

39

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

Paper

40

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

41

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

Sickle Cell disease

42

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

Polyacrylamide gel electrophoresis

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

Sodium dodecyl sulfate (SDS)

44

Separates peptides on the basis of mass alone

SDS page

45

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

Isoelectric focusing gel electrophoresis

46

The pH at which a peptide carries no net electric charge

Isoelectric point

47

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

Two-dimensional Electrophoresis with Isoelectric focusing followed by SDS PAGE

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

Immunoblotting (western blot)

49

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

50

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

X-ray crystallography and NMR spectroscopy

51

NMR is typically only used for the determination of

Small proteins (

52

Which amino acid ridgifies the protein backbone?

Proline (P)

53

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

Arginine, Aspartic Acid, Glutamic Acid, Lysine

54

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

10,000 Daltons

55

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

56

The average weight of an amino acid is

110 Daltons

57

Can form disulfide bonds between their two sulfur atoms

Cysteine pairs

58

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

Disulfide bonds

59

Have no side chains and as a result, are more flexible than other amino acids

-often found in tight turns within a protein

Glycine

60

Often used to make a peptide more rigid

Proline

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