Details About Protein Sequencing Experiment


Protein sequencing is mainly detecting the primary structure of proteins and the amount of the polypeptide chain. In many cases, the polypeptide can be used as proteins. As we all know that the detection of amino acid is the base of the chemistry of proteins research.


The approaches we always take are including the chemical method and enzymatic digestion, and then we can sequence the content and composition of amino acid residues. In this post, I mainly introduce the theory, tips and N-terminal sequencing analysis of protein sequencing.

Tips for sequencing

1. Splitting the polypeptide chain
Proteins, which are constituted by multiple polypeptide chain, should be disassembled. Several polypeptide chains, linked by non-covalent bonds, are called oligomeric protein.

2. Detecting the amount of polypeptide chain in protein molecules
We can determine the amount of the polypeptide chain by detecting the relationship between the moles of terminal amino acid residues and protein molecular weight.

3. Disulfide bond breakage
Polypeptide chains are linked together with the disulfide bond. In order to make the disulfide bond is reduced to a mercapto group, we can processed them in 8mol / L urea or 6mol / L guanidine hydrochloride. And then we can protect the mercapto group by using the alkylating agents from oxidizing again.

4. Detecting the amino acid composition of each polypeptide chain and then calculating molecular ratio in amino acid.

5. Analyzing N-terminal and C-terminal
The amino acid of polypeptide includes N-terminal and C-terminal. N-terminal analysis service is much more important than C-terminal analysis.

6. Polypeptide chains break into several peptide piece.

7. Detecting the order of amino acid of each peptide

8. Determining the order of peptide in the polypeptide chains

9. Determining the location of disulfide bonds.

As we mentioned above, the N-teminal sequencing service is very important. Here I will introduce some precautions of this service.

If we choose the method of Edman degradation method to do the N-terminal sequencing, we will meet some problems inevitably. These problems will affect the protein sequencing a lot.

The first one is the purity of samples. Some experience shows that the sample purity should be over 90%, so it is suitable for the protein sequencing experiment.

The second is the N-terminal blocking and glycosylated protein, which cannot be used to the protein sequencing and it is very difficult to finish the reaction of Edman.

Protein synthesis mainly depends on the N-terminus. So it is very important for the protein sequencing analysis to detect the N-terminal sequencing. And this can be achieved by the methods of Edman degradation and mass spectrometry techniques.

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