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European Biopharmaceutical Review

How Strong is a Peptide Bond?

As a protein chemist and director of a Good Manufacturing Practice-accredited protein sequencing facility, I often encounter a lot of unnecessary and expensive transport and storage conditions of protein samples, shipped to us for the completion of Edman degradation in our facility and often for identification purposes and/or purity checks only.

A lot of companies have these samples shipped in/on dry ice at -70oC, often with a temperature data-logger, special couriers and so on – all expensive and completely unnecessary measures based on non-scientific fairy tales that have unfortunately also been included in several pharmaceutical compendia. Although in some very rare and special cases these low temperatures may help in maintaining the protein’s biological activity, it is entirely nonsense for the purpose of later Edman degradation (1,2,3), for which biological activity is totally irrelevant. After all, the Edman chemistry itself is under very harsh conditions, coupling under an extremely high pH (11-12) and cleavage reaction extreme acidic conditions (100% TFA, pH <1) (1,2,3). Only the protein sequence (either its N-terminus and/or internal sequence) is important, which leads to the final scientific question to address: how strong is a peptide bond? In order to determine proper storage and shipment conditions for these protein samples, this is a question that must be considered.

Bond Strength

A peptide bond is an amide bond: -NH-C=O-, which is a chemically strong bond. Similar bonds are found in Twaron (aramide) – the world’s strongest fibre.

Funnily enough, in the same compendia, it is outlined that it must be boiled in 6MHCl for at least 24 hours, to hydrolyse peptide bonds into their individual amino acids, if you start with a protein. As you can imagine, for many this must mean that peptide bonds are indeed very stable at normal room temperatures, and even at 37oC. You only have to consider your own body temperature to realise that we mainly consist of well-functioning proteins and enzymes.

It is, therefore, entirely incomprehensible that some writers of monographs in pharmaceutical compendia and other non-scientific quality assurance/quality control (QA/QC) individuals maintain the idea that protein samples for Edman degradation need to be stored at least at -20oC. Peptide bonds do not break at room temperature unless you have put the protein in extreme acid or a base solution, which of course nobody does, or unless the protein has autocatalytic enzymatic activity and it was put in a suitable buffer for such enzymatic cuts.

Even if some internal bonds are broken for a relatively low percentage, the sample would be even more suitable for identification by Edman degradation. Good facilities are easily able to interpret multiple signals, if present. After all, all free N-termini will be sequenced: N-terminus plus possible internal sequences at minor level results in even better identification of the protein. If your protein sample was dried down in a speed-vac, it can be safely sent by room temperature to the facility. Forget all the rubbish written in compendia, or the questionable opinions of non-scientific QA inspectors!

Risk Analysis

Now, some of you might think: “What about the risks, if I send protein samples at room temperature?” or “Suppose the postman leaves them lying in the sun?” Inevitably, a courier service is always better if you live in a country where the national post is not so reliable. But if the protein sample was dried properly, and preferably salt-free, there is no chance of any peptide bond cleavage.

Furthermore, you will find that your results do not vary, even if you used to send it at -70oC. The sample will be perfectly suitable for Edman degradation. Just ask the many companies that already utilise this service and have been doing so for a number of years – and of the many samples already analysed, we can easily conclude that we have already (indirectly) validated this assertion. An unexpected or ‘deviation’ result has never been reported due to shipping at room temperature. Show this article to any critical QA inspector, if they ask about this.

The Edman Advantage

How much could you save by evading unnecessarily expensive transportation costs for your company, and not using costly dry ice shipments with temperature data-loggers for each batch release testing for Edman degradation samples?

For identification purposes, the determination of the N-terminal protein sequence is far more specific than intact mass spectrometry (MS) analysis. After all, with MS it is only possible to determine a mass (which may even be a polysaccharide, not a protein). Take a sequence analysis of 15 amino acids N-terminally. In fact, identification of the first 15 amino acids is, in most cases, more than enough for a correct and unique identification of a protein, considering that there are 20 different amino acids possible per residue, implying 2015 = 3.23 x1019 possible different sequences at random. So, if you find the correct sequence, you can be very sure it is the right protein. A performed MS analysis of a peptide map of the protein, which shows correct masses found for all theoretically expected internal peptides, provides additional proof. After all, MS is a good complementary technique, which some people believe will never replace Edman degradation.

Conclusion


Avoid mixing up biological activity with peptide bond strength! For the best results, proceed as above.

References
1. Edman P, Acta Chem Scand 10: pp761-768, 1956
2. Ilse D and Edman P, Aust J Chem 16: pp411-416, 1963
3. Hewick RM, Hunkapiller MW, Hood LE and Dreyer WJ, J Biol Chem 256: pp7,990-7,997, 1981

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Dr Henk J Bak is a biochemist, specialising in protein chemistry and particularly protein sequence analysis. He studied Chemistry at the University of Groningen. After completion of his PhD, Henk worked at the university on a project financed by a third party, which investigated the effect of protein modifications on the functional properties of gluten proteins. At the same time, he founded Eurosequence and since 1987, he has worked as the company’s Managing Director and Chief Scientific Officer.
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