Functional identification of protocadherin alpha 9 (PCDHA9) as a candidate causative gene for amyotrophic lateral sclerosis

ABSTRACT: Mice were sacrificed after anesthesia by isoflurane following neck cutting. The spinal cord L3-L5 was taken and frozen in liquid nitrogen immediately and the ventral horn of the bilateral spinal cord (about 400um thick in gray matter area) was collected at low temperature. The samples were digested with Ripa lysate (containing 4% sodium dodecyl sulfate, protease inhibitors and phosphatase inhibitors). The protein concentrations were detected using a BCA protein assay kit (Thermo Scientific, Rockford, IL). Protein digestion was performed using the filter-aided proteome preparation (FASP) method. After digested with sequencing grade trypsin (Promega, Madison, WI) at 37 °C overnight, the resultant tryptic peptides were labeled with acetonitrile-dissolved TMT reagents (Thermo Scientific, Rockford, IL) by incubation at room temperature in dark for 2 h. The labeling reaction was stopped by 5% hydroxylamine, then equal amounts of labeled samples were mixed before prefractionation with reversed phase (RP)-high performance liquid chromatography (HPLC). Sample prefractionation was performed using an offline basic RP-HPLC approach. The LC-MS/MS analysis was performed using an Orbitrap Fusion™ Lumos™ Tribrid™ mass spectrometer (Thermo Scientific, Rockford, IL Waltham, MA) coupled online to an Easy-nLC 1200 in the data-dependent mode. The database search was performed for all raw MS files using the software MaxQuant (version 1.6). The Mus musculus proteome sequence database downloaded from uniProt (https://www.uniprot.org/) was applied to search the data. The functional results were analyzed by GO analysis.

ORGANISM(S): Mus Musculus

SUBMITTER: Zhiheng Xu  

PROVIDER: PXD042929 | iProX | Mon Jun 12 00:00:00 BST 2023

REPOSITORIES: iProX