Methods in protein sequencing
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Methods in protein sequencing. Example Problem 1. Given an unknown peptide, UkP, determine the sequence from the following data. Amino acid analysis (6N HCl, 24 hrs, 100 o C) gave the results shown. Glu1Ser1Ala2Ile1Tyr1Arg2Met1Val1Gly2Pro1Leu2.

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Methods in protein sequencing

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Methods in protein sequencing

Methods in protein sequencing


Example problem 1

Example Problem 1

Given an unknown peptide, UkP, determine the

sequence from the following data.

  • Amino acid analysis (6N HCl, 24 hrs, 100o C) gave the results shown.

  • Glu1Ser1Ala2Ile1Tyr1Arg2Met1Val1Gly2Pro1Leu2

___-___- ___- ___-___-___-___-___-___- ___-___-___- ___-___-___


Example problem 1 cont

Example Problem 1, cont.

2. Treatment of UkP with a-chymotrypsin gave two

peptides which were purified & analyzed.

C-1 Ala, Arg, Gly, Leu, Ser, Tyr (DNP-Ala)

C-2 Ala, Arg, Glu, Gly, Ile, Leu, Met, Pro, Val (DNP-Gly)

Ala-___- ___- ___-___-Tyr-Gly-___-___- ___-___-___- ___-___-___

3. Treatment of C-1 and C-2, separately with trypsin each

gave two peptides which were purified & analyzed.

C-1:T-1 Leu, Ser, Tyr (DNP-Leu) T-2 Ala, Arg, Gly (DNP-Ala)

Ala-Gly-Arg - ___-___-Tyr-Gly-___-___- ___-___-___- ___-___-___

Ala-Gly-Arg-Leu-Ser-Tyr-Gly-___-___- ___-___-___- ___-___-___


Example problem 1 cont1

Example Problem 1, cont.

Ala-Gly-Arg-Leu-Ser-Tyr-Gly-___-___- ___-___-___- ___-___-___

3. Continued:

C-2:T-3Arg, Gly, Leu, Pro (DNP-Gly) T-4Ala, Glu, Ile, Met, Val (DNP-Val)

Ala-Gly-Arg-Leu-Ser-Tyr-Gly-___-___-Arg-Val-___- ___-___-___

4. Treatment of T-4 with cyanogen bromide gave two

peptides which were purified & analyzed.

T-4:CN-1 Glu, Val, HSerCN-2 Ile, Ala (DNP-ILE)

Ala-Gly-Arg-Leu-Ser-Tyr-Gly-___-___-Arg-Val-Glu-Met-___-___

Ala-Gly-Arg-Leu-Ser-Tyr-Gly-___-___-Arg-Val-Glu-Met-Ile-Ala


Example problem 1 cont2

Example Problem 1, cont.

5. Treatment of CN-2 with hydrazine and analysis gave Ala

as the free amino acid (confirms previous data).

6. Mild acid hydrolysis of the original peptide gave six

small peptides which were purified and analyzed.

A-1 Arg, Leu, Ser, TyrA-5 Ala, Ile, Met

A-2 Arg, Glu, Leu, Val A-6 Arg ,Leu, Gly

A-3 Ala, Gly A-7 Gly, Leu, Pro, Tyr

A-4 Gly, Pro A-8 Glu, Met, Val

Ala-Gly-Arg-Leu-Ser-Tyr-Gly-___-___-Arg-Val-Glu-Met-Ile-Ala

-Arg-Leu-Ser-Tyr-

-Leu-Arg-Val-Glu-

Ala-Gly-

-Gly- Pro-

-Met-Ile-Ala

-Gly-Arg-Leu-

-Tyr-Gly-Pro-Leu-

-Val-Glu-Met-

Ala-Gly-Arg-Leu-Ser-Tyr-Gly-Pro-Leu-Arg-Val-Glu-Met-Ile-Ala


Example problem 1 cont3

Example Problem 1, cont.

Final sequence:

Ala-Gly-Arg-Leu-Ser-Tyr-Gly-Pro-Leu-Arg-Val-Glu-Met-Ile-Ala


Example problem 2

Example Problem 2

Samples of an unknown peptide are subjected to the two

treatments below. After each treatment the resultant

peptides are purified and sequenced by Edman's method

to provide the data given. Determine the sequence of the

unknown peptide using the overlapping peptide method.

1. Cyanogen bromide:2. Trypsin:

Asp-Ile-Lys-Gln-HserGln-Met-Lys

LysGly-Met-Asp-Ile-Lys

Lys-Phe-Ala-HserPhe-Ala-Met-Lys

Tyr-Arg-Gly-HserTyr-Arg

___-___-___- ___-___-___- ___-___-___- ___-___-___- ___-___


Example problem 2 cont

Example Problem 2, cont.

1. Cyanogen bromide:2. Trypsin:

Asp-Ile-Lys-Gln-HserGln-Met-Lys

Lys Gly-Met-Asp-Ile-Lys

Lys-Phe-Ala-Hser Phe-Ala-Met-Lys

Tyr-Arg-Gly-HserTyr-Arg

x

x

x

x

x

x

x

x

Asp-Ile-Lys-Gln-Hser

Lys

Gly-Met-Asp-Ile-Lys

Gln-Met-Lys

Tyr-Arg-Gly-Hser

Lys-Phe-Ala-Hser

Tyr-Arg

Phe-Ala-Met-Lys

Tyr-Arg-Gly-Met-Asp-Ile-Lys-Gln-Met-Lys-Phe-Ala-Met-Lys


Example problem 3

Example Problem 3

Treatment of a peptide with mercaptoethanol

resulted in two fragments whose sequences are

given below. Determine the structure of the original

peptide to include the disulfide bridges.

Ala-Phe-Cys-Met-Tyr-Cys-Leu-Trp-Cys-Asn

Val-Cys-Trp-Val-Ile-Phe-Gly-Cys-Lys


Example problem 3 cont

Example Problem 3, cont.

Treatment of a sample of the original peptide with a-chymotrypsin produced peptides which have the compositions shown (disulfides are still intact):

Ala, Phe

Asn, Cys(2), Met, Tyr

Cys, Gly, Lys

Cys(2), Leu, Trp(2), Val

Ile, Phe, Val

Where should these cleavages occur ?

Ala-Phe-Cys-Met-Tyr-Cys-Leu-Trp-Cys-Asn

Val-Cys-Trp-Val-Ile-Phe-Gly-Cys-Lys


Example problem 3 cont1

Example Problem 3, cont.

Cleavages and disulfide bond connections:

Ala-Phe-Cys-Met-Tyr-Cys-Leu-Trp-Cys-Asn

Val-Cys-Trp-Val-Ile-Phe-Gly-Cys-Lys


End of sequencing

End of Sequencing


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