PXD025207

PXD025207 is an original dataset announced via ProteomeXchange.

Title	An HDX-MS optimised approach to characterise nanobodies as tools for biochemical and structural studies
Description	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.
HostingRepository	PRIDE
AnnounceDate	2024-10-22
AnnouncementXML	Submission_2024-10-22_05:30:38.545.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	John Burke
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	No PTMs are included in the dataset
Instrument	impact HD

Revision	Datetime	Status	ChangeLog Entry
0	2021-04-06 04:13:02	ID requested
1	2021-12-14 12:13:32	announced
⏵ 2	2024-10-22 05:30:41	announced	2024-10-22: Updated project metadata.

Rathinaswamy MK, Fleming KD, Dalwadi U, Pardon E, Harris NJ, Yip CK, Steyaert J, Burke JE, HDX-MS-optimized approach to characterize nanobodies as tools for biochemical and structural studies of class IB phosphoinositide 3-kinases. Structure, 29(12):1371-1381.e6(2021) [pubmed]

10.1016/j.str.2021.07.002;

submitter keyword: HDX-MS, PI3K, nanobody

Dr. John E Burke
contact affiliation	University of Victoria
contact email	jeburke@uvic.ca
lab head
John Burke
contact affiliation	University of Victoria
contact email	jeburke@uvic.ca
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/09/PXD025207

PRIDE project URI

• PRIDE
  1. PXD025207
     1. Label: PRIDE project
     2. Name: An HDX-MS optimised approach to characterise nanobodies as tools for biochemical and structural studies