PXD046995

PXD046995 is an original dataset announced via ProteomeXchange.

Title	Protein binder based IP-MS of SLC12A6 (KCC3) in Hek293WT OE cell lines
Description	Specific protein binders with high affinity, such as antibodies or nanobodies, are scarce for most solute carrier (SLCs) transporters. Moreover, general approaches that can systematically generate binders for multi-transmembrane proteins, such as SLCs, channels or GPCRs, are still missing. Purifying and reconstituting transmembrane proteins in their lipidic environments remain challenging, hence membrane proteins normally act as poor input material for binder generation. To overcome this challenge, we first identified state-of-the-art methods to generate protein binders targeting membrane transporters. Next, we produced full length protein or cell lines as input material for 27 SLCs, which were then processed in the selected binder generation platforms. As a result, we obtained >300 binders for 23 SLCs, 1 SLC failed and 3 SLCs are still in process. Then we established a cell-based validation workflow using immunofluorescent and immunoprecipitation methods to process all obtained binders. To further highlight the suitability of high-performance binders as an important tool for the biomolecular characterization of SLCs, we tested whether three SLC12A6 binders could be used in a quantitative co-immunoprecipitation followed by mass spectrometry (IP-MS) approach. This initial analysis of the IP-MS dataset showed that SLC12A6 specific binders are capable of strongly enriching SLC12A6. We also showed that despite high sequence similarity, the different binders showed different binder to target ratios, indicating different affinities to SLC12A6 in cell lysates. With this work, we were able to generate easily renewable and highly specific binders against SLCs, which will greatly facilitate the study of this neglected protein family.
HostingRepository	PRIDE
AnnounceDate	2024-06-17
AnnouncementXML	Submission_2024-06-17_02:51:22.096.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Fabian Frommelt
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2023-11-15 10:07:13	ID requested
⏵ 1	2024-06-17 02:51:22	announced

10.1016/J.JMB.2024.168665;

submitter keyword: Human, IP-MS, SLC12A6 (KCC3), protein binders

Giulio Superti-Furga
contact affiliation	CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, 1090 Vienna, Austria
contact email	gsuperti@cemm.oeaw.ac.at
lab head
Fabian Frommelt
contact affiliation	CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences
contact email	fabian.frommelt@hotmail.com
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD046995
     1. Label: PRIDE project
     2. Name: Protein binder based IP-MS of SLC12A6 (KCC3) in Hek293WT OE cell lines