FOR CSIR NET/GATE EXAM

• Chapter Name – Biochemistry

• Topic Name- Protein Chemistry (Part II)

Lecture No.- 04 By- Dr. Debasish

1 Protein sequencing

2 Peptide sequencing

3 Practice questions
REAGENTS FOR PROTEIN SEQUENCE DETERMINATION

1. Edman Degradation: for reaction with amino terminus of proteins/ peptides and reduction of peptide by one
residue
2. N-terminal analysis: for reaction with amino terminus of proteins/ peptides, but complete hydrolysis of
peptide required
a. FDNB – Sanger’s Reagent
b. Dansyl chloride and Dabsyl chloride
c. Fluorescamine
d. o-phthalaldehyde
EDMAN DEGRADATION
STEP 1: COUPLING
Phenyl isothiocyanate (PITC) reacts with an α-amino group (or in the case of
prolyl residue with an imino group) at the N-terminal end of the polypeptide
chain, to form a phenylthiocarbamyl derivative of the terminal residue. Basic
conditions are required for this reaction. Clearly, a free α-amino group is
required for this reaction to occur

STEP 2: CLEAVAGE
In the presence of strong acid, cleavage occurs at the first peptide bond,
giving the peptide (minus the first residue) and the liberated first residue as
the anilinothiazolinone (ATZ) form. Once other reactants and products have
been washed away, the shortened polypeptide can be taken through another
round of coupling and cleavage to release the second residue, and so on in
a cyclical fashion. Currently, trifluoroacetic acid (TFA) is used for this
cleavage reaction.
EDMAN DEGRADATION
STEP 3: CONVERSION
The ATZ residue is separated from the peptide by extraction in organic
solvent (ethyl acetate or chlorobutane), and is then converted to a more
stable form to allow better analysis. Conversion to the more stable
phenylthiohydantoin (PTH) form is done in aqueous acid (25% TFA, v/v in
water).

STEP 4: ANALYSIS OF PTH RESIDUES

The PTH residue generated by each cycle of Edman chemistry is typically
identified by chromatography, originally thin-layer chromatography and
latterly reversed-phase high-performance liquid chromatography.
IDENTIFICATION OF N-TERMINAL AMINO ACID

FDNB, Dabsyl Chloride, Dansyl Chloride can specifically react with the N-terminal amino group, and yield
DNB-Amino acid, Dabsyl-Amino acid and Dansyl-Amino acid. These products will eventually be identified by
their corresponding chromatographical properties.
PEPTIDE SEQUENCING

"Bottom-Up" Sequencing- (most common) a. Cleave protein into peptides

b. Send peptides into MS for sequencing

"Top-Down" Sequencing- (difficult but fast) a. Send intact protein into mass spec
b. Fragment & sequence

Why peptides instead of proteins?

1. Increased stability
2. Better solubility
3. Greater sensitivity
4. Easier to sequence if < 20 amino acids
5. Fewer (usually <1) translational modifications/peptide
5. Cheaper instrumentation
(proteins require an FTICR for sequencing)
PEPTIDE SEQUENCING BY MS
PEPTIDE CLEAVAGE SITE BY PROTEASE
PRACTICE QUESTIONS
PRACTICE QUESTION 01

Which of the following fragments will NOT be generated after the CNBr treatment with the given amino acid sequence ?

Asp-Met-Leu-Phe-Met-Arg-Ala-Tyr-Pro-Gly-Asn

a). Arg-Ala-Tyr-Gly-Asn
b). Leu-Phe-Met
c). Asp-Met
d). Asp-Met-Leu-Phe
PRACTICE QUESTION 01: SOLUTION

Which of the following fragments will NOT be generated after the CNBr treatment with the given amino acid sequence ?

Asp-Met-Leu-Phe-Met-Arg-Ala-Tyr-Pro-Gly-Asn

a). Arg-Ala-Tyr-Gly-Asn
b). Leu-Phe-Met
c). Asp-Met
d). Asp-Met-Leu-Phe

Ans. D
PRACTICE QUESTION 02

Following shorter peptide fragments are generated upon treatment with two proteases.
Trypsin cleavages Chymotrypsin cleavages

i) Leu-Glu i) Gln-Ala-Phe
ii) Gly-Tyr-Asn-Arg ii) Asn-Arg-Leu-Glu

iii) Gln-Ala-Phe-Val-Lys iii) Val-Lys-Gly-Tyr

Find out the original polypeptide sequence.

a). Leu-Arg-Gly-Tyr-Asn-Arg-Gln-Ala-Phe-Val-Lys
b). Gln-Ala-Phe-Val-Lys-Gly-Tyr-Asn-Arg-Leu-Glu
c). Gln-Ala-Phe-Asn-Arg-Leu-Glu-Val-Lys-Gly-Tyr
d). Val-Lys-Gly-Tyr-Asn-Arg-Leu-Glu-Gln-Ala-Phe
PRACTICE QUESTION 02: SOLUTION
Following shorter peptide fragments are generated upon treatment with two proteases.

Trypsin cleavages Chymotrypsin cleavages

i) Leu-Glu i) Gln-Ala-Phe
ii) Gly-Tyr-Asn-Arg ii) Asn-Arg-Leu-Glu

iii) Gln-Ala-Phe-Val-Lys iii) Val-Lys-Gly-Tyr

Find out the original polypeptide sequence.

a). Leu-Arg-Gly-Tyr-Asn-Arg-Gln-Ala-Phe-Val-Lys
b). Gln-Ala-Phe-Val-Lys-Gly-Tyr-Asn-Arg-Leu-Glu
c). Gln-Ala-Phe-Asn-Arg-Leu-Glu-Val-Lys-Gly-Tyr
d). Val-Lys-Gly-Tyr-Asn-Arg-Leu-Glu-Gln-Ala-Phe

Ans. B
PRACTICE QUESTION 03
A sample of a peptide of unknown sequence was treated with trypsin; another sample of the same peptide
was treated with chymotrypsin. The sequences (N-terminal to C-terminal) of the smaller peptides produced
by trypsin digestion were as follows:

The sequences of the smaller peptides produced by chymotrypsin digestion were as follows:

Deduce the sequence of the original peptide.

a). Asn—Glu—Ser—Arg—Val—Ile—Trp— Thr—Leu—Met—Ile— Met—Val—Ser—Thr—Lys—Leu—Phe

b). Met—Val—Ser—Thr—Lys— Val—Ile—Trp—Thr—Leu—Met—Ile—Leu—Phe—Asn—Glu—Ser—Arg
c). Met—Val—Ser—Thr—Lys—Leu—Phe—Asn—Glu—Ser—Arg—Val—Ile—Trp—Thr—Leu—Met—Ile
d). None of these
PRACTICE QUESTION 03: SOLUTION
A sample of a peptide of unknown sequence was treated with trypsin; another sample of the same peptide
was treated with chymotrypsin. The sequences (N-terminal to C-terminal) of the smaller peptides produced
by trypsin digestion were as follows:

The sequences of the smaller peptides produced by chymotrypsin digestion were as follows:

Deduce the sequence of the original peptide.

a). Asn—Glu—Ser—Arg—Val—Ile—Trp— Thr—Leu—Met—Ile— Met—Val—Ser—Thr—Lys—Leu—Phe

b). Met—Val—Ser—Thr—Lys— Val—Ile—Trp—Thr—Leu—Met—Ile—Leu—Phe—Asn—Glu—Ser—Arg
c). Met—Val—Ser—Thr—Lys—Leu—Phe—Asn—Glu—Ser—Arg—Val—Ile—Trp—Thr—Leu—Met—Ile
d). None of these
Ans. C
PRACTICE QUESTION 04

A polypeptide is cleaved into peptides by treatment with trypsin and cyanogen bromide, and the peptides are
purified and sequenced. The sequences of the peptides (from N to C-terminus) are shown below.

Based on sequences of the overlapping peptides generated by treatment with trypsin and cyanogen bromide
(shown above), which of the peptides could represent the N-terminus of the polypeptide?
a). T3 b). T4 c). C1 d). C2
PRACTICE QUESTION 04: SOLUTION

A polypeptide is cleaved into peptides by treatment with trypsin and cyanogen bromide, and the peptides are
purified and sequenced. The sequences of the peptides (from N to C-terminus) are shown below.

Based on sequences of the overlapping peptides generated by treatment with trypsin and cyanogen bromide
(shown above), which of the peptides could represent the N-terminus of the polypeptide?
a). T3 b). T4 c). C1 d). C2

Ans. C
PRACTICE QUESTION 05

A peptide, Ala-Arg-Gln-Met-Thr-Trp-Lys-Pro-Val, was digested with trypsin to produce

a). Ala-Arg + Gln-Met-Thr-Trp-Lys + Pro-Val

b). Ala-Arg-Gln-Met + Thr-Trp-Lys-Pro-Val
c). Ala + Arg-Gln-Met-Thr-Trp + Lys-Pro-Val
d). Ala-Arg + Gln-Met-Thr-Trp-Lys-Pro-Val
PRACTICE QUESTION 05: SOLUTION

A peptide, Ala-Arg-Gln-Met-Thr-Trp-Lys-Pro-Val, was digested with trypsin to produce

a). Ala-Arg + Gln-Met-Thr-Trp-Lys + Pro-Val

b). Ala-Arg-Gln-Met + Thr-Trp-Lys-Pro-Val
c). Ala + Arg-Gln-Met-Thr-Trp + Lys-Pro-Val
d). Ala-Arg + Gln-Met-Thr-Trp-Lys-Pro-Val

Ans. D
PRACTICE QUESTION 06
Match column A with column B.

A B
1. Determination of the amino acid sequence of a small peptide. 1. CNBr
2. Identification of the amino-terminal residue of a peptide (of which you have 2. Dabsyl chloride
less than 0.1μg).
3. Reversible denaturation of a protein devoid of disulfide bonds. Which 3. Trypsin
additional reagent would you need if disulfide bonds were present?
4. Hydrolysis of peptide bonds on the carboxyl side of lysine and aromatic 4. Urea, β-ME
residues.
5. Cleavage of peptide bonds on the carboxyl side of methionine. 5. Chymotrypsin
6. Hydrolysis of peptide bonds on the carboxyl side of lysine and arginine 6. Phenyalisothiocyanate
residues.

a). A1-B6, A2-B2, A3-B4, A4-B3, A5-B1, A6-B5

b). A1-B6, A2-B5, A3-B4, A4-B1, A5-B2, A6-B3
c). A1-B6, A2-B2, A3-B4, A4-B5, A5-B1, A6-B3
d). A1-B2, A2-B6, A3-B4, A4-B5, A5-B1, A6-B3
PRACTICE QUESTION 06: SOLUTION
Match column A with column B.

A B
1. Determination of the amino acid sequence of a small peptide. 1. CNBr
2. Identification of the amino-terminal residue of a peptide (of which you have 2. Dabsyl chloride
less than 0.1μg).
3. Reversible denaturation of a protein devoid of disulfide bonds. Which 3. Trypsin
additional reagent would you need if disulfide bonds were present?
4. Hydrolysis of peptide bonds on the carboxyl side of lysine and aromatic 4. Urea, β-ME
residues.
5. Cleavage of peptide bonds on the carboxyl side of methionine. 5. Chymotrypsin
6. Hydrolysis of peptide bonds on the carboxyl side of lysine and arginine 6. Phenyalisothiocyanate
residues.

a). A1-B6, A2-B2, A3-B4, A4-B3, A5-B1, A6-B5

b). A1-B6, A2-B5, A3-B4, A4-B1, A5-B2, A6-B3
c). A1-B6, A2-B2, A3-B4, A4-B5, A5-B1, A6-B3
d). A1-B2, A2-B6, A3-B4, A4-B5, A5-B1, A6-B3

Ans. C
PRACTICE QUESTION 07
A protein has 4 equally spaced chymotrypsin sensitive sites which results in peptide fragments A1, A2,
A3, A4 and A5 upon digestion with chymotrypsin. Peptides A5 and A2, represents N-terminal and C-
terminal fragments respectively. Now, you are asked to synthesize this protein with radiolabeled amino
acids. At time t = 0 you added all the 20 amino acids labelled with 14C and initiated the synthesis. At time
t = 6, full length protein is synthesized. If you stop the synthesis of the protein in time t = 1.5 and digest
the protein with chymotrypsin, which peptide will have maximum 14C label than others?

(a) A3 (b) A1 (c) A5 (d) A2

PRACTICE QUESTION 07: SOLUTION
A protein has 4 equally spaced chymotrypsin sensitive sites which results in peptide fragments A1, A2,
A3, A4 and A5 upon digestion with chymotrypsin. Peptides A5 and A2, represents N-terminal and C-
terminal fragments respectively. Now, you are asked to synthesize this protein with radiolabeled amino
acids. At time t = 0 you added all the 20 amino acids labelled with 14C and initiated the synthesis. At time
t = 6, full length protein is synthesized. If you stop the synthesis of the protein in time t = 1.5 and digest
the protein with chymotrypsin, which peptide will have maximum 14C label than others?

(a) A3 (b) A1 (c) A5 (d) A2

Ans. C
A5 A2

N-TERMINAL C-TERMINAL
PRACTICE QUESTION 08
To sequence a protein with quaternary structure stabilized by disulfide bonds, such as that found in the
immunoglobins (blood antibodies) and insulin, one would first have to

a. do an acid hydrolysis
b. add mercaptoethanol
c. do an alkaline hydrolysis
d. add Dabsyl Cl
PRACTICE QUESTION 08: SOLUTION
To sequence a protein with quaternary structure stabilized by disulfide bonds, such as that found in the
immunoglobins (blood antibodies) and insulin, one would first have to

a. do an acid hydrolysis
b. add mercaptoethanol
c. do an alkaline hydrolysis
d. add Dabsyl Cl

Ans. B
PRACTICE QUESTION 09
The Edman reagent that reacts with polypeptides is

a. phenylisothiocyanate
b. Dansyl Cl
c. dinitrofluorobenzene
d. phenylthiohydantoin
PRACTICE QUESTION 09: SOLUTION
The Edman reagent that reacts with polypeptides is

a. phenylisothiocyanate
b. Dansyl Cl
c. dinitrofluorobenzene
d. phenylthiohydantoin

Ans. A
PRACTICE QUESTION 10
A cylic byproduct is formed during Edman degradation of the protein named

a). Phenylisothiocyanate
b). Thiazoline
c). Phenylthiohydantoin
d). Phenylthiocarbamyl
PRACTICE QUESTION 10: SOLUTION
A cylic byproduct is formed during Edman degradation of the protein named

a). Phenylisothiocyanate
b). Thiazoline
c). Phenylthiohydantoin
d). Phenylthiocarbamyl

Ans. B