1 / 12

Methods in protein sequencing

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. Glu 1 Ser 1 Ala 2 Ile 1 Tyr 1 Arg 2 Met 1 Val 1 Gly 2 Pro 1 Leu 2.

koen
Download Presentation

Methods in protein sequencing

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Methods in protein sequencing

  2. 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. • Glu 1 Ser 1 Ala 2 Ile 1 Tyr 1 Arg 2 Met 1 Val 1 Gly 2 Pro 1 Leu 2 ___-___- ___- ___-___-___-___-___-___- ___-___-___- ___-___-___

  3. 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-___-___- ___-___-___- ___-___-___

  4. Example Problem 1, cont. Ala-Gly-Arg-Leu-Ser-Tyr-Gly-___-___- ___-___-___- ___-___-___ 3. Continued: C-2: T-3 Arg, Gly, Leu, Pro (DNP-Gly) T-4 Ala, 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, HSer CN-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

  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, Tyr A-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

  6. Example Problem 1, cont. Final sequence: Ala-Gly-Arg-Leu-Ser-Tyr-Gly-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-Hser Gln-Met-Lys Lys Gly-Met-Asp-Ile-Lys Lys-Phe-Ala-Hser Phe-Ala-Met-Lys Tyr-Arg-Gly-Hser Tyr-Arg ___-___-___- ___-___-___- ___-___-___- ___-___-___- ___-___

  8. Example Problem 2, cont. 1. Cyanogen bromide: 2. Trypsin: Asp-Ile-Lys-Gln-Hser Gln-Met-Lys Lys Gly-Met-Asp-Ile-Lys Lys-Phe-Ala-Hser Phe-Ala-Met-Lys Tyr-Arg-Gly-Hser Tyr-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

  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. 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

  11. 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

  12. End of Sequencing

More Related