An HDX-MS optimised approach to characterise nanobodies as tools for biochemical and structural studies

ABSTRACT: Given the far-reaching applications of nanobodies, a stream-lined approach to characterizing nanobodies tailored to its intended application is warranted. Camelids can produce a large number of antibodies that can bind to a host of epitopes spanning the entire protein of interest, making identification of binders appropriate to your study slow and cumbersome. To overcome this challenge, we propose the use of hydrogen-deuterium mass spectrometry (HDX-MS) to rapidly characterize nanobody epitopes. HDX-MS is a technique that measures the rate of exchange of amide hydrogens in the protein backbone and can provide valuable insight into interaction surfaces with binding partners. Using the example of the lipid signalling complex PI3K, we employed HDX-MS to characterize the epitopes of a large cohort of nanobodies simultaneously with reasonable accuracy. Some of these nanobodies proved to be extremely useful, stabilizing a flexible region in structural studies and blocking signalling inputs from activating partners in in-vitro kinase assays. These experiments were in turn, instrumental in obtaining the first high resolution structure of a PI3K complex, besides providing novel tools to study PI3K signalling in-vivo.

INSTRUMENT(S): impact HD

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: John Burke  

LAB HEAD: Dr. John E Burke

PROVIDER: PXD025207 | Pride | 2021-09-22

REPOSITORIES: Pride