(M) Peptide Sequencing (LAB) Flashcards by Nathalie Grace Castillo

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

Amino group (NH2)
Carboxylic group (COOH)
R group (side chain)
Hydrogen

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

R group/side chain

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

Dipeptide

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The amino group is known to have what terminal residues?

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The carboxylic group is known to have what terminal residues?

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

N to C (amino to carboxylic)

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

To the left

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

To the right

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

Primary

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

Dehydration (removal of H2O)

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In dehydration, one Hydrogen will come from where?

NH2 (it becomes NH)

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In dehydration, OH will come from where?

COOH (it becomes C=O)

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In sequencing, which abbreviation system is usually used?

Single letter abbreviation

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Primary, Secondary, Tertiary, or Quaternary structures?

Involves the use of Greek letters

Secondary (alpha-helix and beta-pleated sheets)

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Primary, Secondary, Tertiary, or Quaternary structures?

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

Tertiary (deals with interactions between amino acid side chains)

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Primary, Secondary, Tertiary, or Quaternary structures?

Composed of multiple polypeptide chains

Quaternary (evident in sub-units)

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

Primary

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

Protein-digesting enzymes

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

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

Overlapping

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

Cysteine

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

2-mercaptoethanol

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

Fragmentation

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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

Overlapping

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

True

T or F: Large proteins should be sequenced as is

False (reduce into smaller fragments first)

What are the 3 techniques for sequence determination?

1. Edman Degradation 2. Sanger 3. Dansyl chloride

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

Electrophoresis and Chromatography

The Sanger Technique was named after who?

Frederick Sanger

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

Sanger Technique

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

The first one (amino terminal; left-most)

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

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

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

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

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

Labeled

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

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

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

6M of HCl

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

110ºC for 24 hours

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

Hydrolysis

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

True

Fluorine is removed due to what?

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

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

Dinitrophenyl

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

False (1st amino acid only)

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

High-performance liquid chromatography (HPLC)

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

False (disadvantage)

What is the full name of dansyl chloride reagent?

5-dimethylamino-1-naphthalenesulfonyl chloride

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

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

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

False (reverse)

What is the reagent used for the Dansyl Chloride technique?

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

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

Fluorescence

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

True

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

False (fluorescence)

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

Dansyl polypeptide

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

False (dansylamino acid)

Which element attacks the sulfur contained in the dansyl chloride?

Nitrogen

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

False (hydrogen also) P.S. di na daw ie-explain ni ma'am further kasi complex daw siya

The Edman Degradation technique was developed by who?

Pehr Victor Edman

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

Edman Degradation technique

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

What reagent is used in the Edman technique?

Phenylisothiocyanate

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

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

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

Phenylthiocarbamyl derivative (PTC)

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

Sulfur (acts as a nucleophile which creates a 5-membered ring) Nucleophiles form bonds by donating electron pairs

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

Intermolecular interaction

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

Anilinothiazolinone derivative (ATZ)

T or F: PTC is more stable than ATZ

False (reverse)

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

Extraction by ethyl acetate in a low pH environment

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

Phenylthiohydantoin derivative (PTH)

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

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

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

High performance liquid chromatography (HPLC)

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

PTH + name of amino acid e.g. PTH alanine and PTH glycine

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

False (Edman)

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

Before the amino acid (left)

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

After the amino acid (right)

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

Trypsin

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

GI tract

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

Chymotrypsin

This fragmentation agent cuts at the carboxyl side of METHIONINE only

Cyanogen bromide

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

True

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

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

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

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

Overlapping fragments help determine this specific genetic blood disease

Sickle cell anemia

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

Sickle Cell Anemia

The RBCs in sickle cell anemia are shaped like what?

Sickle/Crescent moon

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

Valine

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

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

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

False (it is possible)