ABSTRACT: A549 cells were washed with ice-cold PBS three times and harvested using mechanical scraping. Cell pellets were processed as described previously (Marathe et al., 2021). Briefly, the pellet was re-solubilized in Urea-Thiourea Buffer (6M Urea, 2M Thio-urea in Tris-Cl) containing protease inhibitor cocktail (#4693159001, Roche), phosphatase Inhibitor cocktail 2 (#P5726, Sigma) and phosphatase Inhibitor cocktail 3 (#P0044, Sigma). After protein concentration estimation using BCA (#23225, Pierce), 30ug of proteins from each sample were used for subsequent steps. Samples were reduced with 15mM DTT a 40oC for 60 minutes, followed by alkylation with 15 mM iodoacetamide for 30 minutes at room temperature in the dark. The digestion mixture was then diluted 4× with 50 mM ammonium bicarbonate, and the protein was digested with 1 ?g of MS-grade trypsin (#V5280, Promega) overnight at 37°C. The resulting peptide solutions were cleaned with C18 ZipTips, quantified using the Scopes method (Scopes, 1974), and 700 ng of peptides were injected into Thermo Q-Exactive plus instrument via EasyNLC through C18 PepMap EasySpray RSLC analytical column (#ES903, Thermo). The MS instrument was set to an MS1 resolution of 70,000 and an MS2 resolution of 17,500, with acquisition experiments optimized for 120 min LC gradients.
The MS spectra were analyzed using Proteome Discoverer version 2.2 using the standard LFQ workflows by Thermo Fisher as described previously (Marathe et al., 2021). Briefly, tandem mass spectra were searched against the UniProt human database and reversed sequences, using SEQUEST (Eng, McCormack and Yates, 1994). Full tryptic search with up to 2 missed cleavages and a minimum peptide length of 6 is considered. Carbamidomethylation on cystines (+57.021) was considered as a static modification, and oxidation on methionine (+15.995 Da) was considered as a dynamic modification. Precursor mass tolerance was set to 10 PPM, and fragment mass tolerance was set to 0.02 Da. Precursor ion-based quantification was performed using only unique peptides and the samples were normalized with respect to the total peptide amount. The false discovery rate (FDR) at the protein level was set to 0.01. Three biological replicates were used for analysis, and statistical analysis of identified proteins was performed using ANOVA (background-based).
WT (AN1.raw,BN1.raw,CN1.raw)
KO (AN2.raw,,BN2.raw,CN2.raw)
Rescue (AN3.raw,BN3.raw,CN3.raw)
Prof. S. Murty Srinivasula 