Mass Spectrometry_Sup_Prostate Cancer
Mass Spectrometry was carried out from the cell supernatant. 1. DKD Cell (C4-2 cell Stably knockdown with RB1 and TP53) under control and Neuropilin-2 depleted condition. 2. wild type C4-2B Cell
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Mass-Spectrometry was carried out on LNCaP C4-2B, DKD Scr, and DKD SiNRP2 cells. Cells were grown in a 6-well plate in two different conditions - control and knockdown of NRP2. Cells were transfected and grown in serum-free RPMI media for 48hrs. After 48hrs, cell supernatant/conditioned media (CM) was collected (~2ml), cell debris was removed by centrifuging CM @3000g for 30-mins. RPMI media only was used for background correction. Proteins were purified by acetone precipitation to remove vitamins, choline, and other small molecules contaminants. Mass-Spectrometry analysis was carried out through LC-MS/MS using Thermo Q-Exactive-HF mass spectrometer and a nano RSLC Ultimate 3000 from Dionex. Spectra were processed using Mascot (Matrix Science, London, UK; version 2.6.1) and were subjected to a cutoff of 1% false discovery rate. Spectra were processed by MODIRO ver.1.1 (Protagen, Germany) software (from Proteomics & Metabolomics Facility of the Nebraska Center for Biotechnology at the University of Nebraska, Lincoln).