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BMS > Methods of Protein Analysis > Flashcards

Methods of Protein Analysis Flashcards

1
Q

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

A

Individual amino acid residues

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2
Q

Individual amino acid residues can be:

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

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

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3
Q

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

A

Corresponding Gene

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4
Q

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

A

Peptide Bonds

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5
Q

The sequence of amino acids in a protein is called the

A

Primary Structure

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6
Q

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

A

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

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7
Q

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

A

Peptide Bonds

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8
Q

How can you break a peptide bond?

A
  1. ) Nonenzymically by hydrolysis or by prolonged exposure to a strong acid or base at elevated temp.
  2. ) With an enzyme
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9
Q

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

A

Left

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10
Q

All amino acid sequences are read from the

A

N- terminus to C-terminus

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11
Q

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

A

Peptide bond

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12
Q

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

A

-yl

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13
Q

Isomerically, the peptide bond is typically a

-Due to steric constraints

A

Trans bond

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14
Q

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

A

110°C for 24 hours.

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15
Q

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.

A

cation-exchange Chromatography

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16
Q

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

A

Ionic strength and pH

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17
Q

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.

A

Ninhydrin

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18
Q

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

A

Ninhydrin derivative

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19
Q

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

A

Successive cycles of Edman Degradation

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20
Q

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

A

A sequence of fifteen N-terminal residues

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21
Q

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

A

One- or two-dimensional gel electrophoresis

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22
Q

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

A

N-terminal sequencing

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23
Q

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

A

DNA sequence of the corresponding gene

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24
Q

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

A

Phenylisothiocyanate

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25
Q

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

A

Edman Degradation

26
Q

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

-Used to cleave proteins into smaller peptides.

A

Proteases (peptidases)

27
Q

Two of the most commonly used proteases are the digestive enzymes

A

Trypsin and Chymotrypsin

28
Q

Where does Trypsin cleave?

A

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

29
Q

Where does Chymotrypsin cleave?

A

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

30
Q

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

A

Exopeptidases

31
Q

Cleave within a protein

A

Endopeptidases

32
Q

The movement of charged particles in an electric field

A

Electrophoresis

33
Q

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

A

Electrophoresis

34
Q

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

A

Net charge

35
Q

In Electrophoresis, what is the

  1. ) Negative electrode?
  2. ) Positive electrode?
A

1.) Cathode 2.) Anode

36
Q

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

A

Two-dimensional Electrophoresis

37
Q

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

A

Diagonal

38
Q

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

A

Two-dimensional pattern

39
Q

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

A

Paper

40
Q

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

A

Paper Electrophoresis performed by altering pH to change charge

41
Q

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

A

Sickle Cell disease

42
Q

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

A

Polyacrylamide gel electrophoresis

43
Q

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

A

Sodium dodecyl sulfate (SDS)

44
Q

Separates peptides on the basis of mass alone

A

SDS page

45
Q

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

A

Isoelectric focusing gel electrophoresis

46
Q

The pH at which a peptide carries no net electric charge

A

Isoelectric point

47
Q

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

A

Two-dimensional Electrophoresis with Isoelectric focusing followed by SDS PAGE

48
Q

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

A

Immunoblotting (western blot)

49
Q

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

A

Amino acid sequence and identification of post-translational modifications

50
Q

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

A

X-ray crystallography and NMR spectroscopy

51
Q

NMR is typically only used for the determination of

A

Small proteins (

52
Q

Which amino acid ridgifies the protein backbone?

A

Proline (P)

53
Q

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

A

Arginine, Aspartic Acid, Glutamic Acid, Lysine

54
Q

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

A

10,000 Daltons

55
Q

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

A

30 and 50 angstroms wide

56
Q

The average weight of an amino acid is

A

110 Daltons

57
Q

Can form disulfide bonds between their two sulfur atoms

A

Cysteine pairs

58
Q

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

A

Disulfide bonds

59
Q

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

-often found in tight turns within a protein

A

Glycine

60
Q

Often used to make a peptide more rigid

A

Proline

BMS (62 decks)

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