1. Methods in protein sequencing

2. Example Problem 1 Given an unknown peptide, UkP, determine the sequence from the following data. 1.Amino acid analysis (6N HCl, 24 hrs, 100 o C) gave the results shown. Glu1Ser1Ala2 Ile1Tyr1Arg2 Met1Val1Gly2 Pro1Leu2 ___-___- ___- ___-___-___-___-___-___- ___-___-___- ___-___-___

3. Example Problem 1, cont. 2. Treatment of UkP with  -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 -___-___- ___-___-___- ___-___-___

4. Example Problem 1, cont. 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 -___-___- ___-___-___- ___-___-___ Ala - Gly - Arg - Leu - Ser - Tyr - Gly -___-___- Arg - Val -___- ___-___-___ Ala - Gly - Arg - Leu - Ser - Tyr - Gly -___-___- Arg - Val - Glu - Met -___-___ 4. Treatment of T-4 with cyanogen bromide gave two peptides which were purified & analyzed. T-4:CN-1 Glu, Val, HSer CN-2 Ile, Ala (DNP-ILE) Ala - Gly - Arg - Leu - Ser - Tyr -G ly -___-___- Arg - Val - Glu - Met - Ile - Ala

5. 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 - -Met - Ile - Ala -Leu - Arg - Val - Glu- Ala - Gly- - Gly - Pro - - Val - Glu - Met -- Gly - Arg - Leu -- Tyr - Gly - Pro - Leu - Ala - Gly - Arg - Leu - Ser - Tyr -G ly - Pro - Leu - Arg - Val - Glu - Met - Ile - Ala

6. Example Problem 1, cont. Final sequence: Ala - Gly - Arg - Leu - Ser - Tyr -G ly - Pro - Leu - Arg - Val - Glu - Met - Ile - Ala

7. 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 ___-___-___- ___-___-___- ___-___-___- ___-___-___- ___-___

8. 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 Lys-Phe-Ala-Hser Phe-Ala-Met-Lys Gly-Met-Asp-Ile-Lys Tyr-Arg-Gly-Hser Gln-Met-Lys Tyr-Arg Lys Tyr-Arg-Gly-Met-Asp-Ile-Lys-Gln-Met-Lys-Phe-Ala-Met-Lys Asp-Ile-Lys-Gln-Hser x x x x x x xx

9. 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

10. Example Problem 3, cont. 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 Treatment of a sample of the original peptide with  -chymotrypsin produced peptides which have the compositions shown (disulfides are still intact):

11. Example Problem 3, cont. Ala-Phe-Cys-Met-Tyr-Cys-Leu-Trp-Cys-Asn Val-Cys-Trp-Val-Ile-Phe-Gly-Cys-Lys Cleavages and disulfide bond connections:

12. End of Sequencing