SimPLIT: Simplified Sample Preparation for Large-Scale Isobaric Tagging Proteomics

Large scale proteomic profiling of cell lines can reveal
molecular
signatures attributed to variable genotypes or induced perturbations,
enabling proteogenomic associations and elucidation of pharmacological
mechanisms of action. Although isobaric labeling has increased the
throughput of proteomic analysis, the commonly used sample preparation
workflows often require time-consuming steps and costly consumables,
limiting their suitability for large scale studies. Here, we present
a simplified and cost-effective one-pot reaction workflow in a 96-well
plate format (SimPLIT) that minimizes processing steps and demonstrates
improved reproducibility compared to alternative approaches. The workflow
is based on a sodium deoxycholate lysis buffer and a single detergent
cleanup step after peptide labeling, followed by quick off-line fractionation
and MS2 analysis. We showcase the applicability of the workflow in
a panel of colorectal cancer cell lines and by performing target discovery
for a set of molecular glue degraders in different cell lines, in
a 96-sample assay. Using this workflow, we report frequently dysregulated
proteins in colorectal cancer cells and uncover cell-dependent protein
degradation profiles of seven cereblon E3 ligase modulators (CRL4CRBN). Overall, SimPLIT is a robust method that can be easily
implemented in any proteomics laboratory for medium-to-large scale
TMT-based studies for deep profiling of cell lines.