A systematic comparison of label-free, SILAC and TMT techniques to study early adaption towards inhibition of EGFR signaling in the colorectal cancer cell line Difi

We evaluated the quantification strategies label-free (LF), SILAC and TMT and their performance in quantification of proteins and phosphosites (p-sites) to identify the most powerful approach for monitoring cellular signaling. We analyzed the EGFR signaling network, which plays an essential role in colorectal cancer, and studied its dynamics within 24 h upon treatment with the EGFR blocking antibody cetuximab, representing the first cellular adaption towards therapy. LF achieved a superior coverage, but was outperformed by label-based approaches regarding technical variability, especially for quantification of p-sites. TMT showed the lowest coverage and most missing values. We found that its performance considerably decreases when experimental replicates are distributed over several TMT plexes. SILAC showed the highest precision and outstanding performance for quantification of p-sites, rendering it the method of choice for analyzing cellular signaling in cell culture models. On the protein level, we obse...