Description:
<jats:title>Abstract</jats:title><jats:p>Membrane proteins are an important category of drug targets in human diseases. However, membrane protein characterization is challenging due to their hydrophobicity, low abundance, and poor protease accessibility of the proteins. To facilitate reproducible and sensitive profiling of the druggable membrane proteome, we reported a strategy combining membrane protein extraction and data‐independent acquisition mass spectrometry (DIA‐MS). By a single‐shot DIA analysis for a model lung cancer cell line, the results show good enrichment of 2,740 confidently identified membrane proteins, which accounts for 71% proteins identified from the membrane‐enriched sample and includes 202 FDA‐approved drug targets and 462 uniquely identified membrane proteins compared to the result from whole‐cell lysate. Furthermore, the membrane fraction enrichment significantly enhanced protein abundance of 1,245 membrane proteins, and 22 proteins were identified in the non‐small‐cell lung cancer pathway, including 6 lung cancer drug targets. The approach demonstrated better identification in higher intensities and signal‐to‐noise ratios of peptide ions. The drug targets increased 3.3–25.9‐fold (median 15.5‐fold) in peptide abundance and 1.0–12.0% in (median 5.3%) protein coverage. The membrane proteomic profiling strategy will advance the applications of quantitative proteomics in drug target discovery.</jats:p>