Fig. 4

Proteomics and phosphoproteomics profiling of TRAP-overexpressing cells. Experimental workflow employed to analyze the proteome and phosphoproteome of control (ctrl, Light SILAC labeled) and TRAP3^high (Heavy SILAC labeled) MDA-MB-231 cells (n = 2). Protein extracts were digested with trypsin and then equal amounts of Light and Heavy peptides were pooled. Peptides were fractionated by HiRIEF using immobilized pH gradient (IPG) strips with pH range 3–10 for proteomics analysis, and with pH range 2.5–3.7 for phosphoproteomics analysis, prior to LC-MS analysis (a). Number of unique phospho-peptides identified in each biological replicate, broken down by number of phosphorylations (b). Protein log₂(ratio H/L) (c) and phosphorylation sites normalized log₂(ratio H/L) (d) distribution for the two biological replicates. A threshold for significance was set at −/+3 MAD and −/+ 2.5 MAD away from the median for standard proteomics and phosphoproteomics analysis respectively. “n=” indicates the number of replicates