Mass spectrometry-based proteomic analysis of NSCLC tumor and biopsy samples

The associated publication reports proteogenomic analysis of non-small cell lung cancer, where we identified molecular subtypes with distinct immune evasion mechanisms and therapeutic targets, and validated our classification method in separate clinical cohorts. This protocol describes the sample preparation and mass spectrometry (MS)-based in-depth and rapid proteomic analyses of tumor and biopsy samples. We deployed single-pot solid-phase-enhanced sample preparation (SP3). For the in-depth analysis, we used TMT labeling, followed by high-resolution isoelectric focusing (HiRIEF) prefractionation and LC-MS with data-dependent acquisition (DDA). The reported protocol achieved analytical depth of close to 14,000 quantified proteins and almost 10,000 across the entire cohort of 141 samples. The rapid analysis was label-free, based on LC-MS with data-independent acquisition (DIA). The median number of identified proteins was 3,967 and 3,552 in two independent cohorts of tumor samples (n = 141 and 208, respectively), and 2,494 in another cohort of biopsy material (n = 84).