Serum-free conditioned media (CM) from ATIIER:KRASV12 cells treated without or with 4-OHT (250 nM, 24 hrs) were analysed using an enrichment strategy based upon Strataclean resin (Agilent) in combination with the quantitative label-free approach, LC-MSE, to provide in-depth proteome coverage and estimates of protein concentration in absolute amounts52 (link) (Details provided in Supplementary Methods).
Raw data were processed and collated into a single .csv document. Values were then normalised to total fmol of each sample multiplied by 10,000. Pseudo-counts were applied to the normalised values to replace missing ones, to allow for full statistical analysis to be completed53 (link). We first sorted the normalised values in each column in order of abundance, in ascending order, then the minimum value of each sample identified. This minimum was used to replace all missing values in the data set. A two-tailed, unpaired Student's t-test was used to compare two groups for independent samples. P < 0.05 was considered statistically significant.
In order to highlight their implications in IPF, differentially expressed proteins/genes identified in the quantitative secretome analysis were searched in LGEA web portal (https://research.cchmc.org/pbge/lunggens/mainportal.html) for their levels in pulmonary epithelial cells from control and IPF lung tissue.