The aim of Pure MHC’s research is to discover peptides that are unique-to or only expressed in-diseased cells, or are so highly upregulated that they are druggable targets, giving them disease-specific targets for the development of novel immunotherapies and diagnostics.
They have a unique and patented approach for solubilizing HLA which allows them to make a large amount of HLA for building their de-novo libraries. However, a main challenge of studying HLA, is that all the peptides are biochemically similar, and thus tend to elute around the same time. There are also challenges around reproducibility: CVs tend to be quite high compared to those from tryptic peptides which ionize more reproducibly and are less susceptible to ion suppression.
One way they have overcome these challenges is by using a two-dimensional analytical technique coupled to nanoflow LC-MS. Deploying the new OptiFlow® Turbo V Ion Source on their TripleTOF® 6600+ LC-MS/MS System has provided them the reproducibility and sensitivity they need when working with tissue samples, which are only available in very limited quantities.
Wanting to establish a complimentary absolute quantification workflow, they chose the SCIEX Triple Quad™ 6500+ LC-MS/MS System. They have found the instrument to be very reliable, with the sensitivity they need, and with a large dynamic range of 1x1014 coupled to fast cycle times using Scheduled MRM, they are able to perform absolute quantification on a large number of analytes.
Outcomes of research
- Since upgrading to the OptiFlow® Source a few months ago, they’ve seen a great improvement in reproducibility
- Their CVs are now routinely below 30%, which was not previously the case
- Sensitivity tripled, and they no longer experience ion suppression