Rapid High-pH Reverse Phase StageTip for Sensitive Small-Scale Membrane Proteomic Profiling

Membrane proteins are crucial targets for cancer biomarker discovery and drug development. However, in addition to the inherent challenges of hydrophobicity and low abundance, complete membrane proteome coverage of clinical specimen is usually hindered by the requirement of large amount of starting materials. Towards comprehensive membrane proteomic profiling for small amounts of samples (< 10 µg), we developed high-pH reverse phase (Hp-RP) combined with stop-and-go extraction tip (StageTip) technique, as a fast (~ 15 mins.), sensitive, reproducible, high-resolution and multiplexed fractionation method suitable for accurate quantification of the membrane proteome. This approach provided almost 2-fold enhanced detection of peptides within transmembrane helix domains compared with strong anion exchange (SAX) and strong cation exchange (SCX) StageTip techniques. More than 5000 proteins (63% membrane proteins) can be identified in only 10 µg of membrane protein digests, showing the superior sensitivity of the Hp-RP StageTip approach. The Hp-RP StageTip method enabled in-depth membrane proteome profiling of 11 lung cancer cell lines harboring different EGFR mutation status, which resulted in the identification of 3983 annotated membrane proteins. This provides the largest collection of reference peptide spectral data for lung cancer membrane sub-proteome. Finally, relative quantification of membrane proteins between Gefitinib-resistant and -sensitive lung cancer cell lines revealed several upregulated membrane proteins with key roles in lung cancer progression.