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. How can I then analyze it by Edman degradation / sequencing – and what is causing the problems?

Answer:

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

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

One of the frequent Edman degradation problems is that the N-terminal residue is modified in such a way that it does not react 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 can also occur after isolation during sample manipulation.

These following steps can help prevent this:

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 N-terminal Sequencing and Sequencing Blocked N-terminals.

References:

[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