Application of Edman Degradation N-Terminal Sequencing in Analytical 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 routine method for analyzing this property. Analysis of the 15 amino acid sequence of a N-terminal by Edman degradation is usually a mandatory item in biopharmaceutical declarations. It is also an annual inspection project for many listed biopharmaceuticals). In scientific research, proteins with unknown or uncertain theoretical sequences can be used to provide critical information for confirmation using N-terminal sequencing. Therefore, this method has been widely used in scientific research and industry. What is its principle? How to analyze the results, and what are the precautions for sample storage?

Principle

It can be explained from the following two aspects.

Edman degradation: Through the reaction between PITC (phenyl isothiocyanate) and α-amino group of the protein or N-terminus polypeptide, the first amino acid of the N-terminus is cleavable from the sequence to obtain the free PITC-derived amino acid. At the same time, the α-amino group of the second amino acid residue in the sequence is exposed.

Sequencing: The fragmented PITC-derived amino acids are separated by HPLC and the amino acid species are determined based on the retention time.

With the above two steps, the first amino acid at the N-terminus is analyzed. Sounds complicated? This series of work can be done with automated sequencers, such as PPSQ-31A/33A protein sequencer. Shimadzu is the only company that produces PPSQ-31A/33A in current market.

 Results Analyzing

The automated sequencer ran the experiment quickly, while the results are manually analyzed. So what are the results we usually encounter? (take the first run as an example)

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

Clearly, a peak of Glu (DTT, DMPTU, DPTU are reagent peaks). It is very indicated clearly that the first amino acid at the N-terminus of the sample tested is Glu. Researchers would love to see this result.

However, there are also unusual situations. There are two unusual types:

There are multiple amino acid peaks in the map. This is the case when the sample is not pure enough, or the sample may be degraded (or is not pure enough). Don’t believe it? You may try the sample with HPLC or run an SDS-PAGE gel. You can also use mass spectrometry to measure the molecular weight and see the purity of the sample.)

No peaks of an amino acid at all? There are two reasons for this. N-terminal blocking, ie, the α-amino group of the N-terminal amino acid is modified (eg. acetylation has occurred, Glu is cyclized to pyroglutamic acid, etc.). The amount of loading is too small. The signal strength does not reach the detection line.

Although there are automated sequencers, the speed of N-terminal sequencing is much slower than nucleic acid sequencing. So, how do you make a pre-judgment on your sample before sending it in order to see if it is suitable for analyzing the N-terminal sequence?

Frequently Asked Questions Q&A

Are there requirements of protein sample purity for N-terminal sequencing?

Yes. The purity is preferably above 95%, otherwise there will be multiple amino acid peaks per cycle and cannot be assigned to the protein sequence.

How to analysis N-terminal sequence when there are two or three chains of proteins.

SDS-PAGE electrophoresis is used to separate the bands, and then the proteins on the gel are transferred to the PVDF membrane. After staining with Ponceau, the corresponding bands are cut and sequenced.

There are two tips: Do not use Tris-Glycine buffer when transferring membranes, which will result in a higher background in N-terminal sequencing. CAPS buffer is recommended. Also, when dyeing PVDF membranes, do not use Coomassie Brilliant Blue and should be stained with Ponceau.

What samples are not suitable for analysis of sequences using Edman degradation N-terminal sequencing?

First, the N-terminally closed sample: It is known from the reaction principle that the reaction of the α-amino group at the N-terminus cannot be carried out.

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