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

The N-terminal amino acid sequence of a protein is one of the key quality attributes of a biopharmaceutical. Edman degradation N-terminal sequencing is a conventional method for analyzing this property. Analysis of the 15 amino acid sequences at the N-terminus by the Edman degradation method is usually a mandatory inspection item when applying for biopharmaceuticals. It is also an annual inspection item for many biopharmaceuticals already on the market. In scientific research, N-terminal sequencing can be used for proteins with unknown or uncertain theoretical sequences to provide key information for confirmation. Therefore, this method is widely used in scientific research and industry. While, what is its principle, how to analyze the results, and what are the suggestions for sending samples?

To put it simply, we can understand from these two aspects:

Step I: Edman degradation. The organic reagent of PITC (benzene isothiocyanate) reacts with the α-amino group at the N-terminus of a protein or peptide, so that the first amino acid at the N-terminus is cleaved from the sequence to obtain a free PITC-derived amino acid. At the same time, the α-amino group of the second amino acid residue in the sequence is exposed.

Step II: Sequencing. The separated PITC-derived amino acids are separated by HPLC to determine the type of amino acid according to the retention time.

It completes a cycle and analyzes the first amino acid at the N-terminus. Sounds complicated? This series of work is done by automated sequencers. For example, Shimadzu's PPSQ-31A / 33A protein sequencer is universal and the only one in production on the market.

The automated sequencer finishes the experiment, and the results of this experiment had to be analyzed manually. What results do we usually encounter? (Take the first loop as an example)

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

Clearly, one peak of Glu (DTT, DMPTU, DPTU are reagent peaks). It is very clear that the first amino acid at the N-terminus of the sample being tested is Glu.

However, there are some unusual situations, mainly two types:

There are multiple amino acid peaks in the map. This is what happens when the sample is not sufficiently pure, or if the sample is degraded (or is not sufficiently pure). (Doubtful? You may try one sample with the HPLC, or an SDS-PAGE gel, and you can also use the mass spectrometry to determine the molecular purity.)

No peak of an amino acids at all! There are two reasons for this situation.  N-terminal blocking may occur, that is, the α-amino group of the N-terminal amino acid is modified (such as acetylation modification, Glu cyclization to pyroglutamic acid, etc.). The mount of sample is too little to reach the detection line for the signal.

Although automated sequencers are available, N-terminal sequencing can be much slower than nucleic acid sequencing. Therefore, how to make a pre-judgment of the sample before sending samples to see if it is suitable to use this method to analyze the N-terminal sequence?   

Q&A

Is there a requirement on protein samples for N-terminal sequencing?

Yes. It is best to be above 95%. Otherwise, multiple amino acids will peak each cycle and cannot be assigned to the protein sequence.

How to perform N-terminal sequence analysis if there are two or three strands?

Separated the bands by SDS-PAGE electrophoresis, and then the proteins on the gel are transferred to PVDF membranes. After staining with Ponceau Red, the corresponding bands are cut and sequenced. Do not use Tris-glycine buffer when transferring membranes. It will generate a high background in N-terminal sequencing. CAPS buffer is recommended. In addition, when dyeing PVDF membranes, do not use Coomassie Brilliant Blue. Instead, use Ponceau Red.

What kind of samples are not suitable for sequence analysis by Edman degradation N-terminal sequencing?


The N-terminal blocked sample: From the reaction principle, it can be seen that after the α-amino group at the N-terminal is modified. The reaction cannot proceed.

There are too many non-standard amino acids in the N-terminal sequence of interest. Because there is no corresponding standard, there is no way to analyze by N-terminal sequencing. At this situation, mass spectrometry can be used for sequence confirmation.

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