KCAS Q&A: Immunoaffinity and Hybrid LCMS

Immunoaffinity LC-MS/MS or hybrid LCMS is an approach that has been gaining a lot of traction in the past several years. The technique allows one to quantitate low level analytes (proteins or peptides in many cases) with exquisite selectivity and sensitivity. Many pharmaceutical companies are trying to expand their expertise in this area and develop sensitive assays for both biotherapeutics as well as biomarkers. KCAS is one of the few CROs that can currently support this space.

Below are a few questions asked by customers…


What is Hybrid LCMS or Immunoaffinity LC-MS/MS?

It is a technique that combines an immunocapture step with LCMS.  It is called “Hybrid” because it is a hybrid between a traditional ligand binding assay (LBA) and a mass spectrometry based assay. Typically the target of interest is “captured” either at the protein or peptide level using magnetic beads or column based supports. The analyte (protein) is then digested to produce surrogate peptides which are then specifically detected via an LC-MS/MS method.


How do you know when to use Hybrid LCMS vs. LBA?

This is always a good question. The two approaches are very complimentary and in many cases either approach will give you the answer. However, there are cases where Hybrid LCMS might be a better choice. For example, if you don’t have a great reagent or you have only one reagent, the LCMS approach might be a better solution. Hybrid LCMS only requires one reagent for the initial capture step. The detection on the back end is via the MS.  Furthermore, the initial capture step for the hybrid LCMS approach is only used to help enrich or clean up the sample. This means it does not need to be perfectly selective as you have the very selective MS/MS as your ultimate detector. There are also cases where you have a small sequence change between species which may not be detected in an LBA method but the MS can “dial” in the correct sequence so it could offer better translatability. Lastly, there are examples where there is in interference in the LBA method, that is not problematic in the MS approach.  This is definitely a question that should be discussed at the beginning of a project to help identify the best approach for your specific project.


Can Immunoaffinity LC-MS/MS / Hybrid LCMS help develop an assay when traditional techniques have failed? (SPE, PPT, LBA)

Absolutely, this is the area where the hybrid LCMS approach shines.  Many times the background is just too noisy to be able to get a good clean signal for the analyte of interest.  The key is removing that background.  By using a reagent to enhance the analyte of interest (typically an antibody to the target) you can eliminate the background and enrich for your molecule of interest.  Many times this approach can increase your sensitivity by several orders of magnitude. You can also have multiple steps of enrichment.  For example, you can initially enrich at the protein level and then you can digest the molecule to surrogate peptide and then enrich again at the peptide level.  Every enrichment step needs to be evaluated for the benefits over the losses to make sure the additional step is necessary.


Can Immunoaffinity LC-MS/MS / Hybrid LCMS help understand more information about the molecule that doesn’t show up in traditional LBA methods?

Yes, since you are using a MS detector as the back end, you know the exact sequence and identity of the peptide you are quantitating.  You can easily look at post-translational modifications (assuming they have a different molecule weight/sequence or chromatographic retention time).  You can also use this approach to simultaneously monitor several parts of the molecule if you were concerned about a particular area or stability.  These assays can very easily be multiplexed for many peptides or even proteins depending on your enrichment strategy.


What type of sensitivities can this approach provide?

I would stack up Hybrid LCMS as one of the most sensitive approaches compared to any LBA assay.  At the end of the day sensitivity comes down to signal-to-noise.  The combination of the capture step with the most sensitive MS can provide exquisite selectivities and sensitivities.  In order to get the most sensitive assays, you should also consider micro or nano flow chromatography combined with large injection volumes and multidimensional chromatography. The general approach to get the best sensitivity is to clean it up very selectively (with an antibody), inject as much material as you can, concentrate that on a trap and then introduce it into the most sensitive MS in the narrowest peak (nano or micro LC) possible.


KCAS is one of the very few CROs that can support large molecule LC-MS/MS of which Hybrid LCMS is one emphasis. We have the ability to enrich at the protein or peptide level using bead and column based automation.  We also have microflow multidimensional capabilities for the ultimate in sensitivity.  We can support from early Discovery through to GLP or regulated clinical assays.  Furthermore, we have an excellent LBA group and would love to engage with our clients in delivering the best high level science to address their most challenging issues.