PXD025099

PXD025099 is an original dataset announced via ProteomeXchange.

Title	Facilitating in situ cross-linking and mass spectrometry by antibody-based protein enrichment
Description	Cross-linking of living cells followed by mass spectrometry identification of cross-linked peptides (in situ CLMS) is an emerging technology to study protein structures in their native environment. One of the inherent difficulties of this approach is the high complexity of the samples following cell lysis. This difficulty largely limits the identification of cross-links to the more abundant proteins in the cell. Here, we describe a targeted approach in which an antibody pulls a specific protein-of-interest out of the lysate. Mass spectrometry analysis of the protein material that binds to the antibody can then identify considerably more cross-links on the antibody target and its interactors. By using an antibody against the CCT chaperonin, we obtained over two hundred cross-links that provide in situ evidence for the subunit arrangement of CCT and its main interactions with prefoldin. Antibodies against tubulin likewise provided in situ evidence for the structure of the microtubule including the seam. Finally, the approach was also successful in identifying cross-links on a protein expressing at very low amounts (tau in non-neuronal cells). These results demonstrate the general applicability of antibody-based sample simplification for in situ CLMS.
HostingRepository	PRIDE
AnnounceDate	2022-06-09
AnnouncementXML	Submission_2022-06-09_04:45:53.199.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Joanna Zamel
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Q Exactive HF

Revision	Datetime	Status	ChangeLog Entry
0	2021-03-30 02:46:53	ID requested
⏵ 1	2022-06-09 04:45:54	announced

Kelly JJ, Tranter D, Pardon E, Chi G, Kramer H, Happonen L, Knee KM, Janz JM, Steyaert J, Bulawa C, Paavilainen VO, Huiskonen JT, Yue WW, Snapshots of actin and tubulin folding inside the TRiC chaperonin. Nat Struct Mol Biol, 29(5):420-429(2022) [pubmed]

submitter keyword: Antibody, CL-MS

Nir Kalisman
contact affiliation	Nir Kalisman, Dept. of Biological Chemistry, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel
contact email	nirka@mail.huji.ac.il
lab head
Joanna Zamel
contact affiliation	The Hebrew University of Jerusalem
contact email	joanna.zamel@mail.huji.ac.il
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/2022/06/PXD025099

PRIDE project URI

• PRIDE
  1. PXD025099
     1. Label: PRIDE project
     2. Name: Facilitating in situ cross-linking and mass spectrometry by antibody-based protein enrichment