Sonication-assisted protein extraction improves proteomic detection of membrane-bound and DNA-binding proteins from tumor tissues.

Deep-scale, mass spectrometry-based proteomic studies by the Clinical Proteomic Tumor Analysis Consortium (CPTAC) program involves tissue lysis using urea buffer before data acquisition via mass spectrometry for quantitative global proteomic and phosphoproteomic analysis. This is described in a 2018 protocol(1). Here we report an update to this initial protocol by implementing a sonication step into urea-based tissue lysis. Similar to the initial CPTAC protocol, we identified >12,000 proteins and >25,000 phosphopeptides in a tandem mass tag (TMT) set containing both nonsonicated and sonicated tumor tissues from patient-derived xenograft mouse models. An improvement in the detection of membrane-bound and DNA-binding proteins was observed by including the sonication. We also offer recommendations for optimal sonication conditions such as the buffer composition, timing of sonication cycle, instrumentation settings and a troubleshooting section for potential users. Additionally, the protocol is equally applicable to other biological specimens.