Influencing Factors and Principles of Edman Sequencing

Principle of PTH-Amino Acid Analysing by Edman Sequencing

In general, in each sequencing cycle, the PTH-amino acid is first separated by an Edman sequencer, and then determine the PTH-amino acid chromatogram with HPLC, which is followed by comparison with the standard HPLC chromatograms of 20 amino acids. Finally, the corresponding amino acids information is obtained. The standard HPLC profile of 20 amino acids is shown in the lower panel.


HPLC chromatograms of 20 amino acids

Protein N-terminal Blocking Case

Edman degradation method has been widely used as the gold standard for the N terminal sequence analysis of existing protein samples. However, in practical applications, the Edman degradation method also has certain limitations. For example, one of our customers sent a sample to our library to sequence the N-terminus of a recombinant protein expressed in Bacillus. However, after we transferred the sample to the PVDF membrane, it was found that there was no signal during the sequencing, and the replication turned out the same. Based on our experience, we speculated that the N-terminal blockage may occur in the recombinantly expressed protein.

What is N-terminal Blockage of the Protein?

The n terminal of the protein has a free alpha amino group under unblocked condition. Reaction of the PITC to the alpha amino group is the first step in the Edman protein sequencing reaction. When the N-terminus of the protein is blocked, the α-amino group at the N-terminus is modified so that the N-terminus lacks the free α-amino group, resulting in the fact that the PITC cannot actually bind to the protein so that the Edman degradation reaction cannot proceed. In fact, 50% of the N-terminus of natural proteins has been modified. Common modifications include acetylation, methylation, and pyroglutamination. In addition, sometimes N-terminal blocking modification may also occur during the purification of protein samples. This is mainly due to the reaction between the detergent or chemical substance in the solution and the N-terminal functional group of the protein sample, or the pH level of the solvent used for separation and purification is too high.

At present, the Edman degradation method cannot sequence the protein sample when N-terminal blockage occurred. Under such circumstance, the sample can be cut into peptide fragments by protease and then sequenced using liquid chromatography coupled with mass spectrometry. If the modification that caused the protein N-terminal blockage is known, the corresponding proteinase can be used to cleave the modification of the N-terminus, and then it can be sequenced using Edman degradation reaction. For example, the pyroglutamic acid is cyclized and blocked at the N-terminus of many antibody drugs. After the enzyme is digested with pyroglutaminase, the antibody can be directly sequenced using Edman degradation method. 

Edman protein sequencing

Factors Affecting Edman Degradation Results

If the purity of the chemical reagent used for sequencing is not high enough, there may be a false peak in the PTH chromatogram, which may affect the accurate determination of the amino acid sequence.

Due to the differences of chemical or physical properties of various amino acid residues, the strength of the PTH signal changes while judging its content. For example, tyrosine and serine are prone to dehydroxylation, which may lead to peak change. It is difficult to detect signals when cysteine is modified. Histidine and arginine are difficult to extract because of polarity, and methionine is easy to be oxidized. These factors will lead to a correspondingly lower peak.

The presence of a modified residue with a similar peak to that of other unmodified amino acids can also lead to amino acid misidentification.

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