PXD014216

PXD014216 is an original dataset announced via ProteomeXchange.

Title	Identification of LonB protease interacting proteins in Haloferax volcanii by pull-down assays coupled to MS
Description	In this study, pull down assays combined with mass spectrometry analysis were performed in Haloferax volcanii cells containing different LonB content to identify the repertoire of LonB interacting proteins. H. volcanii H26 and HVLON3 (Lon deficient mutant) were grown in Hv-Min medium and samples were taken at exponential (EXP) and stationary (ST) phase. The cells were treated with crosslinkers DSP (1 mM) for 2 h or formaldehyde (1%) for 10 min at room temperature. Crosslinking reactions were stopped and the cells were harvested, washed, and suspended in saline phosphate buffer containing protease inhibitors. The cells were disrupted, cell debris was removed, and membranes were pelleted and washed in the same buffer. Membranes were incubated in 1× PBS with 1% dodecyl maltoside (DDM) for 1 h at 42 °C and then subjected to immunoprecipitation with anti-NmLon polyclonal antiserum conjugated to Protein A−Sepharose beads CL-4B. As a negative control, in parallel, samples were immunoprecipitated with the non-specific anti-Nep antiserum corresponding to the extracellular N. magadii protease. After incubation with the antibodies, the bead slurry was added to the extracts, gently agitated overnight at 4 °C and washed three times with 1× PBS containing ammonium hydrogen carbonate (AMBIC). On-bead digestion was performed by adding 10 μL of trypsin (10 ng/μL) in 100 mM AMBIC and incubated overnight at 37 °C with agitation. The peptide mix was recovered and 5% (v/v) formic acid was added. The peptides were dried in Speedvac, suspended in 100 μL of Buffer A (0.1% formic acid in water), desalted by zip-tipping (C18 membranes) and analyzed by MS, as described in Cerletti et al. (2018). Proteins were identified and quantified using MaxQuant version 1.5.3.17 with LFQ algorithm. The parameters were set as described in Cerletti et al. (2018); threshold Andromeda scores for unmodified and modified peptides were 0 and 40, respectively.
HostingRepository	jPOST
AnnounceDate	2020-06-11
AnnouncementXML	Submission_2022-09-18_03:12:45.997.xml
DigitalObjectIdentifier
ReviewLevel	Non peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Micaela Cerletti
SpeciesList	scientific name: Haloferax volcanii DS2; NCBI TaxID: 309800;
ModificationList	unknown modification; 2-pyrrolidone-5-carboxylic acid (Gln); L-methionine sulfoxide; deamidated residue
Instrument	LTQ Orbitrap Elite

Revision	Datetime	Status	ChangeLog Entry
0	2019-06-11 06:52:03	ID requested
1	2020-06-10 08:00:05	announced
2	2021-02-04 16:42:52	announced	2021-02-04: Updated FTP location.
⏵ 3	2022-09-18 03:12:46	announced	2022-09-18: Updated FTP location.

Dataset with its publication pending

submitter keyword: Haloferax volcanii, Lon protease, pull-down

Rosana De Castro
lab head
Micaela Cerletti
contact affiliation	IIB-UNMdP
dataset submitter

jPOST dataset URI

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.jpostdb.org/JPST000616/