Mass spectrum analysis, MS, confirming the theoretical molecular weight of the peptide, is the first and foremost tool when it comes to quality control. The idea is that the full length and fully deprotected peptide can be said to be present in the sample if its mass is found. Furthermore, if the signal obtained is far stronger than other signals, it would indicate that the peptide makes up the majority of the material that is subjected to the analysis. Typically the accuracy of an ESI-MS system is of the order of 0.1%. This is good enough for discriminating between the target peptide and most impurities such as peptides that are not fully deprotected or peptides that are not of full length.
However, in many cases this is not 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. If you want to verify the presence of the S-S bond by MS, then you need the better accuracy of a high end system. When one or two Da could mean everything for the successful outcome of your project, we think that you should be able to verify this in the MS.
A high end MS system is not only more accurate, it also has a better resolution. That means mass verification an be made on mono-isotopic masses. The MS data can then be treated as being semi-quantitative and be used as a second opinion on the purity of the material.
While MS does verify the theoretical molecular weight, indicating that you have the correct peptide, it is certainly no proof of this. A scrambled sequence would of course yield the same result. The only way to confirm sequence integrity is to sequence the material. In MS/MS the peptide is fragmented and the resulting fragments are then identified to verify consistency with the target peptide sequence. We think that you would always benefit from the certainty that this analysis will provide, and especially when you are investing in high purity “mission critical” peptides.
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.
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. We think that although it might not be possible to obtain a certain peptide at the desired purity, you should at least know about it.
Since 1992, Innovagen has been a provider of molecular biology services and research materials for the scientific community. Our extensive experience from thousands of antibody and peptide projects is the basis for our dedication to excellent quality and customer service.
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