ABSTRACT: Two C57Bl6/J male mice were sacrificed by injecting 200 microliters of the lethal anaesthetic Dolethal. Kidneys were perfused with Dulbeccos Phosphate Buffered Saline, dissected, decapsulated and weighted. Kidneys were decellularized using 3 freeze-thaw cycles (freezing at -80C, 30 minute long thawing at 4C) and a 3-day long incubation in 2% SDS at 4C under gentle rotation. Ten to twenty milligram tissue net weight of each sample was homogenised. Protein concentration was determined using Bradford Assay. Ten micrograms of total protein was loaded in SDS-PAGE and migrated for 25min and prepared using the GeLC-MS method (doi: 10.1007/7651_2017_76).
For LC-MS/MS analysis a Dionex Ultimate 3000 HPLC nanoflow system was used in combination with a Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer. Samples were reconstituted in 25 microliters mobile phase A (98% water, 2% acetonitrile, 0.1% formic acid) and 10 microliters were loaded onto the LC column configured with a Dionex 0.1 x 20 mm, 5 micrometres, 100 A C18 nano trap column with a 5 microliters/min flow rate. An Acclaim PepMapTM C18 100 nanoViper column 75 micrometres x 50cm, 2 micrometres 100 A was used with a flow rate of 300nL/min. Peptides were eluted under a 250min gradient from 2% mobile phase B (0.1% formic acid in 80% acetonitrile) to 95% mobile phase B. Voltage of the positive ion electrospray ionization was set to 2.1kV. The mass spectrometer parameters were set as follows: 1) full MS: resolution of 70,000, automatic gain control (AGC) target at 1e6, maximum injection time (IT) of 100ms, scan range of 380 - 1,200m/z; 2) dd-MS2: resolution of 17,500, AGC target at 1e5, maximum IT of 50ms, TopN of 20, normalized collision energy of 30; 3) dd settings: intensity threshold 1.6e5, Minimum AGC target 8.00e3, dynamic exclusion for 15s. Raw files were processed with Thermo Proteome Discoverer 2.4 software, utilizing the Sequest search engine and the UniProt Mus musculus fasta database containing only canonical sequences (downloaded on 31/05/2023, including 17,155 reviewed entries). Carbamidomethylation of cysteine was set as a static modification, and oxidation of methionine and oxidation of proline were set as dynamic modifications. Two missed cleavage sites, a precursor mass tolerance of 5ppm, and fragment mass tolerance of 0.05Da were allowed. Feature mapper node was run with default settings.
Jean-Sebastien Saulnier-Blache 
