Written by Merel Dagher (PEACE Advisor)
Determine the sequence of this hexapeptide: L, K, G, M, S, P. Follow the sequence of clues mentioned below to determine the sequence.
a. First, I use Sanger Sequencing to get a change from FDNB to P-DNB
b. Second, I cleaved with Trypsin to get a dipeptide of the sequence G-M and a tetrapeptide (P, S, K, L)
c. Lastly, I cleaved with Pepsin to get a dipeptide of sequence P-S and a tetrapeptide (K, L, G, M)
a. First, I use Sanger Sequencing to get a change from FDNB to P-DNB
b. Second, I cleaved with Trypsin to get a dipeptide of the sequence G-M and a tetrapeptide (P, S, K, L)
c. Lastly, I cleaved with Pepsin to get a dipeptide of sequence P-S and a tetrapeptide (K, L, G, M)
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The sequence is PSLKGM. The first step tells me that the N-terminus amino acid is Proline. The second step tells me I have a dipeptide and a tetrapeptide. Trypsin cleaves at Lysine and Arginine at the carboxy-terminus. I know my tetrapeptide contains Proline, so it must come before my dipeptide. I know that I cleaved at Lysine, so my 4th amino acid is K. Finally I know the order of my dipeptide, so my sequence so far is P _ _ K G M. Finally Pepsin cleaves at Leucine, Phenylalanine, Tyrosine, and Tryptophan at the amino-terminus. I have a dipeptide of P-S, and since proline is in it, I know that S is my second amino acid. Pepsin cleaved at L at the amino-terminus, which means my third amino acid is Leucine. So my sequence is P S L K G M.