Application of Edman Degradation N-terminal Sequencing in Analyzing Protein and Peptide Sequences

The N-terminal amino acid sequence of proteins is one of the key quality attributes of biopharmaceuticals. N-terminal sequencing of Edman degradation method is a conventional method for analyzing this attribute. The analysis of 15 amino acid sequences at the N-terminal by Edman degradation method is usually a necessary item in the application of biopharmaceuticals. It’s also an annual inspection program for many of the biological drugs that have been put on the market. In scientific research, N-terminal sequencing can be used to provide key information for the identification of proteins with unknown or uncertain theoretical sequences. So this method is widely used in scientific research and industry. So this method is What is the principle of N-terminal Edman degradation? How do we analyze the results and what are some of the points for attention?

What is the principle of N-terminal Edman degradation?

Simply speaking, we can understand this in the following two steps:

The first step was Edman degradation. Through the reaction of PITC (phenylisothiocyanate), an organic reagent, with the alpha-amino group at the N-terminal of a protein or a polypeptide, the first amino acid at the N-terminal is broken off from the sequence to obtain a free PITC-derived amino acid, and the second amino acid residue in the sequence is exposed.

The second step was protein sequencing. The PITC derived amino acids were separated by HPLC and the kinds of amino acids were determined according to the retention time.

The above two steps completes a cycle which could find out the first amino acid of N terminal. Does it sounds complicated? However, this series of work has been done by automated sequencer. The following machine is the PPSQ-31A/33A type protein identification of Shimadzu which is on the market now and the only one in production.

How to do the interpretation of result?

Although the automated sequencer ran through the experiment, the results had to be manually analyzed. So what do we usually get?Let us take the first loop as an example.

Usually for the protein identification service, the sample with a purity of more than 95%, the sequencing results are like the following:

It is very clear that there are only one Glu peak. DTT DMPTU and DPTU as reagent peak. So the result could show very clearly that the N-terminal first amino acid of the detected sample is Glu. And that is the best result that we would like to see. However, there are also unusual situations as follows:

There are multiple peaks of amino acids in the atlas. The reason on this occurred is that the sample is not pure enough, or sample is degraded. All in all, when this kind of case occurred, the reason is lack of purity. If you do not believe that, you can run a sample of HPLC, run a SDS-PAGE glue or you can also use mass spectrometry to determine the molecular weight of the sample to see the purity of the problem.

However, the pure sample sometimes happened that it is very clear on its diagram. There is not a peak of amino acids. There are two reasons for this. One is the N-terminal closure is the modification of the α -Amino of N-terminal amino acids such as the acetylated modification occurred, Glu is cycled to pyroglutamic acid and so on. The other is the sample is too little leads that the signal strength does not reach the detection line. After the above example analysis, if you meet these cases in next time, you would know why will the results happened like this.

Although there are automated sequencers, N-terminal Sequencing is much slower than nucleic acid sequencing. So, how to prejudge your sample before you send it to figure out whether it is appropriate to use this method to analyze N-terminal sequence？

What is the frequency question?

Does N-terminal Sequencing require Purity of protein samples？

Yes. The purity should be more than 95%. Otherwise, each cycle has multiple peaks of amino acids, which can not be assigned to the protein identification service.

How do you do N-terminal sequence analysis if the protein has two or three strands?

The bands were separated by SDS-PAGE electrophoresis and then transferred to the PVDF membrane. The corresponding bands were cut and sequenced. Do not use Tris-glycine buffer during membrane transfer which will has a higher background in N-terminal Sequencing so that CAPS buffer is recommended. In addition, for PVDF membrane staining, do not use Coomassie brilliant blue, it is suggested to use spring red staining.

What kind of sample is not suitable for Edman degradation N-terminal Sequencing to analyze the sequence?

Firstly, when we meet N-terminal sealed sample, from the principle of the reaction, if the N-terminal α -amino was modified, the reaction could not be carried out.

Secondly, there are too many nonstandard amino acids in N-terminal sequence. Because there is no corresponding standard, there is no way to analyze through N-terminal Sequencing. At this time, mass spectrometry can be used to confirm the sequence.