Practice of N-terminal Sequencing by Edman Degradation Technology in Protein and Peptide Sequence Analysis

The N-terminal amino acid sequence of protein is one of the key quality attributes of biological drugs. N-terminal sequencing by Edman degradation is a routine method to analyze this property. The analysis of 15 amino acid sequences on the N-terminal by Edman degradation method is usually a necessary item when applying for biological drugs. At the same time, it is also an annual inspection item of many listed biological drugs (see Pharmacopoeia 2015). In scientific research, N-terminal sequencing can be used to provide key information for the confirmation of unknown or uncertain theoretical sequences. So this method is widely used in scientific research and industry. What are its principles? How to analyze the results? Advice for sample delivery?

Principles

In short, we can understand it from these two aspects:

The first step is Edman degradation. Through the reaction of PITC (Phenylisothiocyanate), an organic reagent, and α - amino acid at the N-terminal of protein or peptide, the first amino acid at the N-terminal is broken from the sequence to obtain free PITC derived amino acid, and the α - amino acid of the second amino acid residue in the sequence is exposed at the same time.

The second step is sequencing. The amino acids derived from the broken PITC are separated by HPLC, and then the amino acids are determined according to the retention time.

All these work is completed by automatic sequencer. For example, the PPSQ-31a/33A protein sequencer of Shimadzu is the only one that is currently in production.
PPSQ-31a/33A protein sequencer (Shima dzu)

Result Analysis

The automatic sequencer finishes the experiment, and the results have to be analyzed manually. So, what results do we usually encounter? Let’s take the first cycle as an example.

Generally, for a protein sample with a purity of more than 95%, the sequencing results are as follows:

 One clesr peak of Glu. DTT, dmptu and DPTU are reagent peaks. It is very clear that Glu is the first amino acid on the N-terminal of the sample. This is the best result you may want to see.

However, there are also unusual situations as follows.


There are many peaks of amino acids in the map. This is the case when the purity of the sample is not enough, or the sample is degraded (or purity is not enough). Son’t believe it? Take a sample and take it to HPLC, or take an SDS-PAGE gel. You may also measure the molecular weight by mass spectrometry to see the purity of the sample.
Completelly clean with no peak of amino acid! There are two reasons for this result. One reason is that the N-terminal is blocked i.e. the α - amino acid of N-terminal amino acid is modified (such as acetylation, Glu cyclization to pyroglutamate, etc.). The other reason is that the amount of sample is too small that the signal strength can not reach the detection line.

Q&As

Q: Are there requirements for the purity of protein samples in n terminal sequencing?

A: Yes. The purity is better to be over 95%, or otherwise, there are more than one amino acid peak in each cycle, which can not be classified to the protein sequence.

Q: When the protein has two or three chains, how to analyze the N-terminal sequence?
A: Firstly, SDS-PAGE is used to separate the bands, and then the protein on the gel is transferred to PVDF membrane. After Ponceau staining, the corresponding bands are cut and sequenced.

There are two points for attention: do not use Tris glycine buffer during membrane transfer, which will produce a high background in N-terminal sequencing. It is recommended to use caps buffer. In addition to PVDF membrane dyeing, do not use Coomassie brilliant blue, but use Ponceau staining instead.

Q: What samples are not suitable for N-terminal sequencing by Edman degradation method?

A: N-terminal blcked samples: from the reaction principle, it can be seen that after the N-terminal α - amino is modified, the reaction can not be carried out.

Samples with too many non-standard amino acids in the N-terminal sequence. Because there is no corresponding standard, there is no way to analyze them by N-terminal sequencing. At this condition, mass spectrometry can be used for sequence confirmation instead.

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