Early in the development stages of any protein therapeutic, a peptide mapping experiment is undertaken. A peptide map involves breaking the macromolecular protein structure down into its constituent peptides for subsequent analysis. Post-analysis, a picture of the protein is formed by piecing the peptides back together, a bit like a jigsaw. This so-called ‘bottom-up’ approach to the characterization of proteins is fundamental because it reveals details about the protein structure on the amino acid level, which is not always possible when looking from the ‘top-down’ at the intact protein.
Every peptide mapping experiment starts in the same place- digestion of the protein. This might sound simple, I mean, we digest proteins in our gut without even thinking about it every single day, so how hard can it be? In theory, it’s not so hard. But in practice, to do it reproducibly and within a time pressured environment, day in, day out, it can be.
Proteins have a number of structural levels that first need to be broken down:
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- Primary – the amino acid sequence
- Secondary – α helices and β sheets involving hydrogen
- Tertiary – 3D structure involving bonding interactions
between amino acid side-chains, e.g. disulphide bridges
- Quaternary – protein subunit interactions to form larger
aggregate protein complexes
There are various established methods of breaking down protein structure, or denaturing a protein, as it is known. Detergents and chaotropes are often employed to do this chemically, followed by reduction of disulphide bonds and alkylation of the free sulphydryl groups to ensure that disulphides do not re-form. All this requires a lot of reagent preparation and optimization, before even considering which enzyme to use to digest the unfolded protein, what conditions are best for the digest (amount of enzyme, temperature, time) and how best to analyse the resultant peptides.
In an earlier blog post one of my colleagues mentioned an Olympic-standard speedy digestion kit that removes a lot of the pain points and irreproducibility associated with traditional in-solution protein digests…
Join me on my visit to the Thermo Fisher Scientific
Chromatography & Consumables Center to find out
more about the latest advances in protein digestion
and discover enzymatic digest tools that could aid
the development of your next blockbuster: