4 reasons to perform N-terminal protein sequence analysis by Edman

Alphalyse
Apr 26. 2017

Question:

How do I obtain the N-terminal sequences of the heavy chain and light chain of my monoclonal antibody? Is there a prefered protein sequence analysis method for mAbs?

Answer:

mAb N-terminal sequence analysis is often performed using high resolution mass spectrometry for peptide mapping by LC MS/MS [1]. However, sequencing by the classical Edman degradation method offers some advantages over LC MS peptide mapping [2]:

  • It determines the exact N-terminus of the heavy chain and the light chain.
  • The termini of recombinant mAbs may be longer than expected, due to partial incomplete removal of leader sequences. This may be observed if the recombinant cell line has been under stress to produce very high levels of protein. Sometimes truncated forms lacking amino acids can also be observed by Edman sequencing.
  • The Edman reaction is performed directly on the HC or LC molecule. This is in contrast to peptide mapping, which you perform on a complex mixture of peptides after proteolytic cleavage.
  • The Edman protein sequence analysis will distinguish between the isobaric amino acids I/L based on chromatographic retention time. Whereas they have identical Mw’s in the mass spectrometry analysis, so you cannot tell them apart.

How do you prepare your samples for protein sequence analysis?

A typical sample preparation of a monoclonal antibody for Edman sequencing follows these steps:

  1. First, reduce the mAb and separate the HC and LC by 1D SDS PAGE. This analysis also shows if the sample is heterogeneous, by showing multiple gel bands.
  2. Then electroblot onto a PVDF membrane, followed by staining of the protein bands by Coomassie or Ponceau.
  3. Next step is to cut the HC, LC bands from the PVDF membrane.
  4. Finally, perform Edman sequencing of 10 cycles, i.e. 10 residues, of both the heavy chain and the light chain PVDF bands. [3]

Protein sequence analysis on the N- and C-terminal

Be aware of issues with sequence analysis due to blocked N-terminal

It is common that the first amino acid in the LC and HC sequence is either a glutamate (Glu) or glutamine (Gln) residue that has undergone cyclization to pyroglutamic acid (pyroGlu).

The cyclization used for protein sequence analysis is a non-enzymatic chemical reaction and is also often partial. Unfortunately, cyclic modification prevents coupling of the PITC reagent and as a result blocks the Edman sequencing reaction.

>> Read more about Edman degradation in this blog post: Why is my protein’s n-terminal blocked? >>

The solution is to treat the PVDF membrane by pyroglutamate aminopeptidase, which removes the pyroGlu . This will de-block the protein for the Edman chemistry to work [3].

Should you separate by HPLC or 1D SDS PAGE?

You may also use HPLC instead of 1D SDS PAGE for separation of HC/LC. However, this sometimes causes problems because the reduced HC/LC often precipitates. Furthermore, you risk that small amounts of antibody remain linked as one HC+LC, thus complicating the sequence interpretation [3].

The solution is to separate the chains by 1D SDS PAGE. Alternatively, you can try sequencing the intact ab with the HC and LC together [3].

Would you like help with protein sequence analysis – or supplementary techniques for mAb characterization?

Alphalyse can help you with your protein sequence analysis. We also offer mAb characterization in more detail:

References:
[1]          Pham et al: High-throughput protein sequencing.“, Analytical Chemistry2003

[2]          Bandeira et al: Beyond Edman Degradation: Automated De novo Protein Sequencing of Monoclonal Antibodies“, Nature Biotechnology2008

[3]          Liu et al: In vitro and in vivo modifications of recombinant and human IgG antibodies”, mAbs, 2014

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