Why is my protein’s N-terminal blocked (and what can I do about it)?

Question:

I found that the N-terminal of my protein is blocked. So, how can I analyze it by Edman degradation / n-terminal sequencing – and what causes the problems?

Answer:

To sequence the protein N-terminal is a requirement according to the ICH Q6B Guideline. This includes structural characterization of recombinant proteins for clinical testing, and demonstration of comparability and consistency between cGMP batches [1].

50% of proteins run into Edman degradation problems

It is estimated that almost half of all proteins cannot be directly sequenced by Edman degradation – because of problems with a blocked N-terminal residue. Thus, you obtain no sequence data if the N-terminus is blocked – either naturally or as a result of sample processing [2].

One of the frequent Edman degradation problems is modification of the N-terminal residue in such a way that it no longer reacts with the Edman reagent phenyl isothiocyanate. For example, the blocked N-terminal residue may be an N-acetyl amino acid. It could also be a glycosylated amino acid, or a pyrrolidone carboxylate group [2, 3].

Of these, you encounter proteins with an acetylated amino acid most frequently. Evidence suggest that about 80% of the soluble proteins in mammalian cells have acetylated N-terminal amino acids [2].

Examples of covalently modification of the N-terminal amino group of a polypeptide

• acetylation − C( = O) − CH₃ : Eliminates the positive charge on the N-terminal amino group by changing it to an acetyl group.
• formylation − C( = O)H : The N-terminal methionine usually found after translation has an N-terminus blocked with a formyl group.
• Pyroglutamate:  An N-terminal glutamine can attack itself, forming a cyclic pyroglutamate group.

N-terminal block also occurs after isolation during sample manipulation

These following steps can help prevent Edman degradation problems:

1. Allow the gel to polymerize well. You need to do this since the free acrylamide might alkylate some amino acids.
2. Use a reducing agent in the electrophoresis running buffer. (Either sodium thioglycolate (0.1 to 1 mM) or 10 mM reduced glutathione).
3. Avoid addition of acetic acid in your staining buffer [3].

Learn more about Protein Sequencing and Sequencing Blocked N-terminals on the Alphalyse website.

References:

For more information about Edman degradation and possible problems, we recommend the following articles:

[1]          European Medicines Agency: “ICH Topic Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products“, 1999

[2]          Wellner et al: “Sequencing of peptides and proteins with blocked N-terminal amino acids: N-acetylserine or N-acetylthreonine“, Proceedings of the National Academy of Sciences of the United States of America, 1990

[3]          Fowler et al: “Removal of N‐Terminal Blocking Groups from Proteins“, Current Protocols in Protein Science, 1996