Question

Explain the process of how a protein is sequenced from start to finish. In your answer, clearly identify the role of 2-mercaptoethanol, dansyl chloride, phenylisothiocyanate, and trifluoroacetic acid.

Answer 1

All the mentioned chemicals are used in different chemical methods of protein sequencing. Protein sequencing refers to the method of determining the amino acid sequence of all or part of a protein or peptide. There are two direct methods of protein sequencing: a) mass spectrometry and b) Edman degradation. Mass spectrometry is the most widely used method of amino acid sequencing and identification but Edman degradation remains a valuable tool for identification of N-terminal amino acid of the peptide or protein.

The usual strategy for determining the amino acid sequence of a protein involves following basic steps.

1. Multimeric proteins (more than one polypeptide chain) are separated into their constituting chains by exposure to low pH, high concentration of urea or guanidinium hydrochloride and purified.
2. Intrachain S--S (disulfide) cross-bridges between cysteine residues in the polypeptide chain are cleaved. If these disulfides bonds are present between two different chains then step 2 precedes step1.
3. Each polypeptide chain is cleaved into smaller fragments.
4. The N-terminal and C-terminal residues are identified.
5. The amino acid composition of each polypeptide chain is determined.
6. Sequence determination of peptide fragments.
7. The overall amino acid sequence of the protein is reconstructed from the sequences in overlapping fragments.
8. The positions of S--S cross-bridges formed between cysteine residues are located.

Edman degradation (N-terminal amino acid analysis):

Phenylisothiocyanate (PITC) or Edman's reagent is used to selectively label the N-terminal amino acid. PTH reacts with an uncharged N-terminal amino group, under mildly alkaline conditions, to form a cyclical phenylthiocarbamoyl derivative. Anhydrous trifluoroacetic acid (TFA) is used to cleave the terminal amino acid in the form of a thiozolinone derivative leaving the other peptide bonds intact. The thiazolinone amino acid is then selectively extracted into an organic solvent (e.g. N-butyl chloride) and treated with mild acid (e.g. 25% TFA/water) to form the more stable phenylthiohydantoin (PTH)- amino acid derivative that can be identified by using either chromatography or electrophoresis. The procedure can be repeated to identify the next amino acid.  Now if a disulfide bond is encountered in the sequence, PTH-cysteine derivative will form and remain attached with polypeptide and PTH-cystein will not be released. This necessitates the reduction of disulfide bond in the polypeptide sequnce before sequencing. Reduction of free cysteine can be done by use of 2- marcaptoethanol. In order to prevent the reoxidation of free sulfahydryl groups (of cysteine) generated after treating with 2-marcaptoethanol, peptide fragments are treated with iodoacetic acid or acrylonitrile which results in the formation of alkyl derivative of the cysteine residue attached with chain. Other method of prevention of forming disulfied bonds is irreversible oxidation of disulfide bond is use of preformic acid which oxidises cysteine to negatively charge cysteic acid and due to repulsion of negatively charged cysteic acid group re-formation of disulfide bonds does not occur. Moreover, in this process alkylation is also not required.

Sanger method of N-terminal amino acid analysis:

Sanger method also uses the same principle of end-labeling and detection but with a different reagent, fluoro-dinitrobenzene (FDNB) called Sanger's reagent. The labeled protein is then hydrolyzed by 6M HCl at 110 ºC. The polypeptide on reaction with FDNB gives a coloured dinitrophenol labeled amino terminal amino acid derivative, which can be identified by its characteristic migration rate on thin-layer chromatography, paper electrophoresis or by absorbance.

Sanger degradation has disadvantage where the N terminal residue can only be determined and all other residues will be hydrolyzed. Thus we cannot repeat the cycle with same sample. On the other hand, these dyes selectively labels the amine groups present in the protein and therefore can label the amine groups present in the side chains as well, which may give erroneous results.

Next comes the dansyl chloride (1-dimethyl aminophthalene-5-sulfonyl chloride) which also labels the N-terminal amino acid and the stable blue- or blue-green–fluorescent sulfonamide adducts. Acidic hydrolysis liberates all amino acid and the N-terminal dansyl amino acid and are separated which can be easily identified by absorbance.

C-terminal amino acid analysis:

This is done by Hydrazine method and carboxypeptidase method.

Hydrazine method (Akabori method) involves heating the protein with annhydrous hydrazine which transform all but C-terminal amino acid into hydrazides. they aere then seperated from the original free C-terminal amino acid. There are four types of Carboxypeptidase, Type A, B, C and Y. Carboxypeptidase A (from bovine pancreas) works well in hydrolyzing the C-terminal peptide bond of all residues except proline, arginine, and lysine. carboxypeptidase B, is effective only when Arg or Lys are the C-terminal residues. Thus, a mixture of carboxypeptidases A and B liberates any C-terminal amino acid except proline. Carboxypeptidase C and Y acts on any amino acid.