My monoclonal antibody is not 100% pure, what is the purity required for de-novo sequencing?
We typically observe two types of protein impurities in antibody samples for de-novo sequence analysis.
If the sample is contaminated with other antibodies it can impact the success rate for a de-novo sequencing project. When the light and heavy chain are digested with several individual proteases and the LC MS/MS peptide data are used to collect the antibody sequence, it is not really possible to tell if a given peptide sequence comes from one or the other antibody. Thus the antibody should be a purified monoclonal containing only 1 antibody.
If the samples contains other proteins, it is in many cases be able to remove these proteins by chromatography or SDS PAGE before the digestion of the HC and LC.
Estimate the purity of your antibody by running an SDS PAGE gel under reducing conditions. This should provide you with two intense bands at around 25 kDa and 50 kDa. Furthermore, you will only get very weak bands from other protein impurities.
It can be difficult from the SDS PAGE to see if the sample contains more antibodies. This is because their LC and HC will also migrate around 25 kDa and 50 kDa. In this case, you can perform LC-MS intact mass analysis of the reduced antibody to see if the sample contains multiple Mw-forms of the HC and LC.
Get help with your de-novo analysis
If you don’t feel confident in performing the purity test yourself, have a look at the Alphalyse Antibody de-novo sequencing webpages.
You can also download our application note for more information about de-novo sequencing with examples of coverage maps.