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biochem > (M) Peptide Sequencing (LAB) > Flashcards

(M) Peptide Sequencing (LAB) Flashcards

1
Q

A chiral alpha carbon has 4 unique linkages, what are those 4?

A
  • Amino group (NH2)
  • Carboxylic group (COOH)
  • R group (side chain)
  • Hydrogen
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2
Q

This component in a chiral carbon defines the shape of the amino acid

A

R group/side chain

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3
Q

What is formed when 2 amino acids are linked together by a peptide bond?

A

Dipeptide

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4
Q

The amino group is known to have what terminal residues?

A

N

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5
Q

The carboxylic group is known to have what terminal residues?

A

C

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6
Q

The direction of the peptide chain runs from and to what terminus?

A

N to C (amino to carboxylic)

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7
Q

The amino group lies where in respect to the alpha carbon?

A

To the left

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8
Q

The carboxylic group lies where in respect to the alpha carbon?

A

To the right

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9
Q

The peptide sequencing is represented by what structure of a protein?

A

Primary

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10
Q

To form a peptide bond, one COOH group and one NH2 group need to react via a process of what?

A

Dehydration (removal of H2O)

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11
Q

In dehydration, one Hydrogen will come from where?

A

NH2 (it becomes NH)

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12
Q

In dehydration, OH will come from where?

A

COOH (it becomes C=O)

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13
Q

In sequencing, which abbreviation system is usually used?

A

Single letter abbreviation

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14
Q

Primary, Secondary, Tertiary, or Quaternary structures?

Involves the use of Greek letters

A

Secondary (alpha-helix and beta-pleated sheets)

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15
Q

Primary, Secondary, Tertiary, or Quaternary structures?

Deals with the types of bonds present (hydrogen bonds, Van der Waals, disulfide bonds, and hydrophobic interactions)

A

Tertiary (deals with interactions between amino acid side chains)

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16
Q

Primary, Secondary, Tertiary, or Quaternary structures?

Composed of multiple polypeptide chains

A

Quaternary (evident in sub-units)

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17
Q

Sequence and amino acid preparation are already easier with this specific type of protein structure because they are already LINEAR

A

Primary

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18
Q

Each polypeptide chain is cleaved into smaller fragments by what enzymes?

A

Protein-digesting enzymes

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19
Q

Polypeptide chains are cleaved if the number of amino acids exceeds how many?

A

50

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20
Q

The overall amino acid sequence is reconstructed from the fragments using what technique?

A

Overlapping

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21
Q

The positions of disulfide cross bridges in this amino acid are located by biochemists

A

Cysteine

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22
Q

This compound can be used to separate chains, destroy disulfide bridges, and make proteins linear

A

2-mercaptoethanol

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23
Q

The process of cleaving polypeptide chains into smaller segments is called?

A

Fragmentation

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24
Q

This process aids in determining the sequence of amino acids as it mostly happens when it is exposed to 2 different reagents causing separate fragmentations

A

Overlapping

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25
Q

T or F: Fragmentation can be done by enzymes or chemical agents capable of cleaving peptides at specific sequences

A

True

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26
Q

T or F: Large proteins should be sequenced as is

A

False (reduce into smaller fragments first)

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27
Q

What are the 3 techniques for sequence determination?

A
  1. Edman Degradation
  2. Sanger
  3. Dansyl chloride
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28
Q

Further purification of fragments can be done by what 2 processes?

A

Electrophoresis and Chromatography

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29
Q

The Sanger Technique was named after who?

A

Frederick Sanger

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30
Q

This technique can determine which amino acid is the first one in the sequence (amino-terminal)

A

Sanger Technique

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31
Q

The Sanger Technique is only able to identify which amino acid?

A

The first one (amino terminal; left-most)

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32
Q

What is Sanger’s reagent that reacts with the N-terminal residue under basic conditions?

A

2,4-dinitrofluorobenzene (DNFB) or
1-flouro-2,4-dinitrobenzene (FDNB)

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33
Q

T or F: DNFB reacts with the free NH2 group of the N-terminus of the last amino acid

A

False (1st amino acid; an N-terminus on the last amino acid doesn’t exist)

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34
Q

When a reagent attaches to the polypeptide chain, the polypeptide molecule is considered to be what?

A

Labeled

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35
Q

T or F: After Sanger’s reagent has attached to the 1st amino acid, it has now been isolated and identified

A

False (you need to break down/remove the other amino acids because the technique only identifies the first)

36
Q

Removal of other non-labeled amino acids in Sanger’s technique requires the usage of what compound?

37
Q

The use of 6M of HCl in Sanger’s technique must be done under what temperature and time duration?

A

110ºC for 24 hours

38
Q

The use of 6M of HCl in Sanger’s technique promotes what biological process of breaking down water?

A

Hydrolysis

39
Q

T or F: Hydrolysis breaks peptide bonds and forms individual amino acids

40
Q

Fluorine is removed due to what?

A

Nucleophile attacks (ma’am did not further explain due to complexity)

41
Q

In Sanger’s technique, a yellow-colored derivative of the DNFB reagent is formed, what is this derivative?

A

Dinitrophenyl

42
Q

T or F: In Sanger’s technique, only the first and second amino acids will be labeled and determined by chromatography

A

False (1st amino acid only)

43
Q

What kind of chromatography determines the first amino acid in Sanger’s technique?

A

High-performance liquid chromatography (HPLC)

44
Q

T or F: The advantage of Sanger’s technique is that it is able to only determine the first amino acid in a chain

A

False (disadvantage)

45
Q

What is the full name of dansyl chloride reagent?

A

5-dimethylamino-1-naphthalenesulfonyl chloride

46
Q

What is the similarity between the Sanger technique and the Dansyl Chloride technique?

A

It reacts with the N-terminal residue of the first amino acid under basic conditions

47
Q

T or F: The Sanger technique is more sensitive than the Dansyl Chloride technique

A

False (reverse)

48
Q

What is the reagent used for the Dansyl Chloride technique?

A

Dansyl Chloride (short name) or 5-dimethylamino-1-naphthalenesulfonyl chloride (full name)

49
Q

The amino acid where Dansyl Chloride is attached uses what detection technique?

A

Fluorescence

50
Q

T or F: Dansyl chloride uses hydrolysis to remove amino acids

51
Q

T or F: Detection of the first amino acid in the Dansyl Chloride technique uses chromatography

A

False (fluorescence)

52
Q

When dansyl chloride combines with a polypeptide chain, it reacts with the N-terminus and therefore it will be called as?

A

Dansyl polypeptide

53
Q

T or F: The separated amino acid that the dansyl chloride is attached to during fluorescence is called a dansyl polypeptide

A

False (dansylamino acid)

54
Q

Which element attacks the sulfur contained in the dansyl chloride?

55
Q

T or F: In the dansyl chloride technique, only chlorine is removed

A

False (hydrogen also)

P.S. di na daw ie-explain ni ma’am further kasi complex daw siya

56
Q

The Edman Degradation technique was developed by who?

A

Pehr Victor Edman

57
Q

This technique can label and cleave peptides from the end terminal without disrupting the peptide bond and breaking the amino acids

A

Edman Degradation technique

58
Q

The Edman technique can sequence peptides of up to how many amino acids?

59
Q

What reagent is used in the Edman technique?

A

Phenylisothiocyanate

60
Q

What is the similarity of the Edman technique to the other 2 techniques?

A

The reagent combines with the N-terminus of the first amino acid under basic conditions

61
Q

What derivative is formed when Phenylisothiocyanate combines with the first amino acid?

A

Phenylthiocarbamyl derivative (PTC)

62
Q

Under acidic conditions of the Edman technique, what molecule in the phenylthiocarbamyl derivative attacks the carbonyl carbon of the first amino acid?

A

Sulfur (acts as a nucleophile which creates a 5-membered ring)

Nucleophiles form bonds by donating electron pairs

63
Q

What kind of molecular interaction happens when sulfur attacks the carbonyl carbon of the first amino acid in the Edman technique?

A

Intermolecular interaction

64
Q

The first amino acid cleaved in the Edman technique is called?

A

Anilinothiazolinone derivative (ATZ)

65
Q

T or F: PTC is more stable than ATZ

A

False (reverse)

66
Q

How is ATZ removed from the chain in the Edman technique?

A

Extraction by ethyl acetate in a low pH environment

67
Q

ATZ is converted to what final derivative in the Edman technique?

A

Phenylthiohydantoin derivative (PTH)

68
Q

What is the order of produced derivatives in the Edman technique?

A
  1. Phenylthiocarbamyl (PTC)
  2. Anilinothiazolinone (ATZ)
  3. Phenylthiohydantoin (PTH)
69
Q

What technique is used to identify the PTH residue in each cycle of the Edman technique?

A

High performance liquid chromatography (HPLC)

70
Q

PTH residues, once attached and isolated to an amino acid, can be named by?

A

PTH + name of amino acid

e.g. PTH alanine and PTH glycine

71
Q

T or F: The remaining peptides can be isolated and subjected to the next degradation cycle in the Sanger technique

A

False (Edman)

72
Q

If a reagent cuts at the amino side, then it cuts at what location with respect to the amino acid?

A

Before the amino acid (left)

73
Q

If a reagent cuts at the carboxyl side, then it cuts at what location with respect to the amino acid?

A

After the amino acid (right)

74
Q

This fragmentation agent cuts AFTER the amino acid (carboxyl side) of basic amino acids (i.e. lysine and arginine)

75
Q

Trypsin can be found in what system of the human body?

76
Q

This fragmentation agent cuts at the carboxyl end of aromatic amino acids (i.e. tryptophan, phenylalanine, and threonine)

A

Chymotrypsin

77
Q

This fragmentation agent cuts at the carboxyl side of METHIONINE only

A

Cyanogen bromide

78
Q

T or F: Sequencing can commence once amino acids are cut

79
Q

Overlap and identify the original sequence of the following sequences exposed to 2 different fragmenting agents (from the internet):

  1. GWR
  2. YL
  3. DS
  4. LD
  5. GW
  6. RY
A

GWRYLDS (glycine, tryptophan, arginine, tyrosine, leucine, aspartic acid, and serine)

80
Q

Overlap and identify the original sequence of the following sequences exposed to 2 different fragmenting agents (from transes):

  1. GGWAK
  2. SFVR
  3. VRGGW
  4. SF
  5. AK
A

SFVRGGWAK (serine, phenylalanine, valine, arginine, glycine, glycine, tryptophan, alanine, and lysine)

81
Q

Overlapping fragments help determine this specific genetic blood disease

A

Sickle cell anemia

82
Q

This is a genetic disease wherein the RBCs cannot carry oxygen efficiently

A

Sickle Cell Anemia

83
Q

The RBCs in sickle cell anemia are shaped like what?

A

Sickle/Crescent moon

84
Q

In sickle cell anemia, there is a change in amino acid sequence, an example of this is when glutamine is replaced with?

85
Q

T or F: Peptide sequencing can tell you if defects are present within a person’s DNA

A

True (it also checks for the arrangement of the sequence)

86
Q

T or F: It is currently impossible to replace amino acid residues with another one to correct the sequence

A

False (it is possible)

biochem (31 decks)

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