3. A solution contains a mixture of three unknown human proteins B-nd which are

Question

3. A solution contains a mixture of three unknown human proteins B-nd which are run on the 2D gel shown on the right a. Estimate the molecular masses and isolelectric points of A, B and C based on their position on the gel (3 pts). b. In order to identify the proteins based on the sequence of the human genome, the three protein spots are cut out from the gel, the proteins are subjected to trypsin cleavage and the resulting peptide mixture is extracted from the gel and subjected to tandem mass spec analysis. Please explain the principle of this method and how it can deliver sequence information that will allow the identification of the proteins, considering that the complete human genome sequence is known (2 pts). 4. A protein biochemist attempted to determine the amino acid sequence of a decapeptide. Use the results from the trypsin, chymotrypsin, and cyanogen bromide treatments to suggest the amino acid sequence of this decapeptide. Answer the following questions (31-35) based on the results below: Trypsin digestion gave three fragments containing the following residues (not in order) T1: Pro, Arg, Phe, Ala T2: Thr T3: Met, Trp, Met, Lys, Va Chymotrypsin digestion gave three fragments containing the following residues (not in order): CT1: Trp, Arg CT2: Pro, Phe, Ala CT3: Met, Thr, Val, Met, Lys Treatment with fluorodinitrobenzene, which labels the N-terminal residue, resulted in an Ala derivative. The cyanogen bromide treatment gave three fragments containing the following residues (not in order): CNBr1: Met, Val CNBr2: Ala, Phe, Pro, Trp, Met, Arg CNBr3: Thr, Lys

Explanation / Answer

3a-

The molecular mass of protein A is around 30kDa and its pI is around 4

The molecular mass of the protein B is around 80kDa and its pI is around 6

The molecular mass of the protein B is around 60kDa and its pI is around 7.5

B- In mass spectroscopy charged particles are moved in the magnetic field. These charged particles deflect in the magnetic field according to their molecular weight. To produce charged particles the compound is first bombarded with the electrons. This charged particles then undergo fragmentation. This will give us the charged particles in the gaseous phase. Finally, all these particles are detected on the basis of their charge to mass ratio and their relative abundance, this ratio and relative abundance help us to give a spectrum. The final spectrum is analysed and protein sequence is determined.

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