on 06-19-202002:42 PM - edited on 10-15-202110:53 AM by Closed Account
Kara Zehr1; Bhavin Patel2; Amarjeet Flora2; Penny Jensen2; Sergei Snovida2; Ryan Bomgarden2; Kay Opperman2; John C Rogers2 ASMS 2020 Purpose: To optimize a protocol for extracting proteins from FFPE tissues compatible with MS sample prep and LC-MS/MS analysis.
Method: We evaluated different paraffin removal protocols and lysis/homogenization techniques using FFPE sections from normal and tumor breast, lung, and colon tissues. We assessed protein yield to identify the most optimized protocol and verified compatibility with Thermo Scientific™ EasyPep™MS sample prep kits, Thermo Scientific™ TMT10plex™ reagents, Thermo Scientific™ Pierce™ High pH Reversed-Phase Peptide Fractionation Kit and subsequent LC-MS/MS analysis. Fresh frozen tissues were compared to FFPE samples.
Results: Higher protein yield was observed using the EasyPeplysis buffer and micro pestle-based tissue homogenization. This optimized method coupled to EasyPepMS sample prep kits allowed identification and quantitation of 2,000-3,500 proteins from breast/lung/colon tissue samples. TMT10plex reagents were used for relative quantification of 5,000 proteins to identify differentially expressed proteins.
1 University of Illinois at Urbana-Champaign, Champaign, IL
2 Thermo Fisher Scientific, Rockford, IL