We deliver high quality peptides. Well, that is an easy thing for us to say, but how do you know whether or not this is true? Not that easy, and if you don’t have expert knowledge on the subject it is virtually impossible to determine. In the below we’re discussing how quality has to be considered in all aspects of the production, the hallmarks of a high quality source, and the tell-tale signs of substandard vendors.
Like cooking a good meal, it starts with using raw materials of high quality – certain Swiss and American sources tend to be of preference. As a trivial example, using cheap sources for reagents is dicey as they are typically not concerned with whether or nor a certain percentage of their amino acids lack the FMOC protection. That means that you get sequences that contain double amino acids. One might think that it is not a problem since the peptide is purified in the end, but the fact of the matter is that these errors might not be all that easy to get rid off – if at all. We will come to this.
High quality reagents are essential, and as it turns out, so is a simple trick like having an acetylation cycle for each amino acid coupling step, capping incomplete sequences. It won’t matter how good you are, there will always be a certain percentage for each amino acid coupling reaction for which the reaction did not complete – the amino acid is “deleted”. If you add the next amino acid at this point then part of the material will have the previous amino acid missing. These are called deletion sequences. On the other hand, if an acetylation cycle is run in between the two reactions, then the failed sequences are effectively terminated and will no longer take part in the synthesis. Again, one might think that it is not a problem since the peptide is purified in the end, but this is not the whole story.
A preparative HPLC is used in the production to purify the peptide. The principle is that the set of physiochemical properties of the full length peptide is unique in this context and that it will separate from the impurities in this process. However, when the impurities are much alike the target peptide in this respect, separation may not be possible. It is often the case with peptides that should be produced at high levels of purity that these need to be subjected to several rounds of purification, where conditions are adjusted in order to achieve the level of separation required. A more discriminating column would improve on resolution and make the separation easier, but it would also mean that the capacity dwindles to a point where it is no longer a viable option.
So, let's say that you want to make a 20-mer peptide, and you have a problem at AA #10. With no capping cycle, you get a deletion sequence, and so some of the material will be 19-mers, with the error at #10. With a capping cycle on the other hand, the peptide is cut off at #10 and you have some material that is 10-mers. It is more likely that you will have a problem sorting out the 19-mer deletion sequence compared to the 10-mer truncation, even with very fancy HPLC equipment. Here we also see the benefit of using high quality reagents, because it is simply not true that these errors always can be remedied by HPLC purification as the resolution of any preparative HPLC is limited.
For quality control however, resolution is important while capacity on the other hand is not an issue. Thus the analytical HPLC should have a far more discriminating column compared to the one used in the preparative HPLC. Depending on what column is used for the HPLC analysis, you will get a different answer to what the purity of the peptide is. If you use an inferior column, the resolution is poor, and the purity shown may be far from the true value. Contaminants are more likely to co-elute with the target. You’ll see this as broad peaks, peaks with shoulders, or the use of steep gradients. Conversely, if you use a highly discriminating column, the resolution is excellent with sharp peaks using a shallow gradient. Thus you are more likely to spot any issues, and you have a chance to attempt to remedy this with additional purification rounds.
Continuing on the topic of quality control; The MS analysis, confirming the theoretical molecular weight of the peptide, is included in any a standard QC setup. Interestingly enough, the accuracy of an MS system is typically in the order of 0.1%. It sounds great, and it is good enough for discriminating between the target peptide and most impurities such as peptides that are not fully deprotected. However, when you consider that 0.1% is roughly 2 Da for a 20-mer peptide, it is clear that in many cases this level of accuracy should not be considered to be sufficient. For example, deamidation of Gln, which is a common error, will result in a mass shift of only 1 Da, and will be overlooked in a standard MS setup. For peptides that contain a disulfide bridge, two hydrogen atoms are lost in the formation of the bond causing a mass shift of 2 Da. Thus, 1-2 Da can be the difference between a biologically active peptide and one that is not.
There is an added benefit to using a better MS system; The results can be treated as being semi-quantitative. If there is a major contaminant co-eluting with the target peptide in the HPLC analysis, then it will show up as a strong peak in the MS. Having found a hidden contaminant makes it possible to re-purify the material in order to obtain truly pure peptide material.
Just as the HPLC results can be made to look better by using a steep gradient, MS results can sometimes be said to confirm theoretical mass by taking of the average of the recorded average masses. Mass spectrum analysis is a great tool, but you want the better accuracy of a high end system that is capable of verifying mono isotopic masses.
Confirming target molecular weight using MS analysis is only indicative of the presence of the correct peptide. It is not proof, since a scrambled sequence would of course yield the same result. The only way to confirm sequence integrity is to sequence the material. If you want the certainty of knowing what you have - and considering that you’re investing in reagents that are absolutely critical for your experiment, you probably do - then MS/MS tandem sequencing of the material is a good idea.
In summary of the above, we can say that everything is connected. The validity of the reported level of purity and the recorded molecular weight is only as good as the hardware will allow for – and only when used with the aim of finding errors. And so, inferior QC will mask poor quality and conversely, if you are using state of the art quality control, then you have to think quality in all steps of the production.
How do you know which lab will supply you with high quality? Look for those with high QC standards; Purity determination using highly discriminating analytical HPLC columns with a shallow gradient, and MS verification using high end equipment on mono isotopic molecular masses.
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