PXD040867-2

PXD040867 is an original dataset announced via ProteomeXchange.

Title	Rat Cortical Primary Neuronal Culture with NRN1 Protein Treatment
Description	Cortical rat neuronal culture, lysis and proteolytic digestion Primary rat cortical neurons were generated from E18 Sprague-Dawley rat embryos with minor modifications (Swanger, Mattheyses et al. 2015, Henderson, Greathouse et al. 2019). Neurons were cultured at a density of 4x105 cells per well in 12-well culture plates (Fisher Scientific, catalog no. 353043). Neurons were cultured in Neurobasal medium (Fisher Scientific, catalog no. 21103-049) supplemented with B27 (Fisher Scientific, catalog no.17504-044). Culture maintenance included a half media change every 2-3 days. At DIV 14, neurons were either treated with 150 ng/mL recombinant NRN1 protein (Abcam, ab69755) or vehicle treated with diH2O for 6 hours. NRN1 concentration was chosen based on published data that identified a plateau in exogenous NRN1 induced effects on transient potassium currents at 150 ng/mL (Yao et al., 2012). After 6 hours neurons were washed 2x with 1 mL 1X phosphate-buffered saline (PBS). To harvest cells, 1 mL 1X PBS + protease inhibitor (Fisher Scientific, catalog no. 78426) was added and cells were centrifuged for 2300rpm for 5 minutes at 4°C. Cell pellets were lysed in 200uL 8M urea buffer and HALT protease and phosphatase inhibitor cocktail (1x final concentration). Lysates were sonicated with a probe sonicator 3 times for 10 s with 10 s intervals at 30% amplitude and cleared of cellular debris by centrifugation in a tabletop centrifuge at 18,000 rcf for 3 minutes at 4° C. Protein concentration was determined by BCA assay and one-dimensional SDS-PAGE gels were run followed by Coomassie blue staining as quality control for protein integrity and equal loading before proceeding to protein digestion. Protein homogenates (50 µg) were diluted with 50 mM NH4HCO3 to a final concentration of less than 2 M urea and then treated with 1 mM dithiothreitol (DTT) at 25°C for 30 minutes, followed by 5 mM iodoacetimide (IAA) at 25°C for 30 minutes in the dark. Protein was digested with 1:100 (w/w) lysyl endopeptidase (Wako) at 25°C for 2 hours and further digested overnight with 1:50 (w/w) trypsin (Pierce) at 25°C. Resulting peptides were desalted with a Sep-Pak C18 column (Waters) and dried under vacuum.
HostingRepository	PRIDE
AnnounceDate	2023-11-14
AnnouncementXML	Submission_2023-11-14_09:03:39.851.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Eric Dammer
SpeciesList	scientific name: Rattus norvegicus (Rat); NCBI TaxID: 10116;
ModificationList	monohydroxylated residue; deamidated residue; iodoacetamide derivatized residue
Instrument	Orbitrap Eclipse

Revision	Datetime	Status	ChangeLog Entry
0	2023-03-14 11:31:54	ID requested
1	2023-07-20 11:57:58	announced
⏵ 2	2023-11-14 09:03:40	announced	2023-11-14: Updated project metadata.
3	2024-10-22 05:56:53	announced	2024-10-22: Updated project metadata.

Hurst C, Pugh DA, Abreha MH, Duong DM, Dammer EB, Bennett DA, Herskowitz JH, Seyfried NT, Integrated Proteomics to Understand the Role of Neuritin (NRN1) as a Mediator of Cognitive Resilience to Alzheimer's Disease. Mol Cell Proteomics, 22(5):100542(2023) [pubmed]

submitter keyword: rat primary cortical neurons,NRN1, neuroproteomics

Dr. Nicholas T. Seyfried, DPhil
contact affiliation	Professor, Emory University School of Medicine Departments of Biochemistry and Neurology
contact email	nseyfri@emory.edu
lab head
Eric Dammer
contact affiliation	Emory University
contact email	edammer@emory.edu
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/2023/07/PXD040867

PRIDE project URI

• PRIDE
  1. PXD040867
     1. Label: PRIDE project
     2. Name: Rat Cortical Primary Neuronal Culture with NRN1 Protein Treatment