title: Rapid High-pH Reverse Phase StageTip for Sensitive Small-Scale Membrane Proteomic Profiling.
abstract: 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. Toward 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 min.), 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 encompassing transmembrane helix (TMH) domain, as compared with strong anion exchange (SAX) and strong cation exchange (SCX) StageTip techniques. Almost 5000 proteins (∼60% membrane proteins) can be identified in only 10 μg of membrane protein digests, showing the superior sensitivity of the Hp-RP StageTip approach. The method allowed up to 9- and 6-fold increase in the identification of unique hydrophobic and hydrophilic peptides, respectively. 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 subproteome. Finally, relative quantification of membrane proteins between Gefitinib-resistant and -sensitive lung cancer cell lines revealed several up-regulated membrane proteins with key roles in lung cancer progression.
authors.affiliation: Institute of Chemistry, University of the Philippines , Diliman Quezon City 1101, Philippines.|Institute of Chemistry, Academia Sinica , Taipei 115, Taiwan.
authors.forename: Baby Rorielyn T
authors.initials: BR
authors.lastname: Dimayacyac-Esleta
authors.affiliation: Institute of Chemistry, Academia Sinica , Taipei 115, Taiwan.
authors.forename: Chia-Feng
authors.initials: CF
authors.lastname: Tsai
authors.affiliation: Institute of Chemistry, Academia Sinica , Taipei 115, Taiwan.|Department of Chemistry, National Tsing Hua University , Hsinchu 30013, Taiwan.|Molecular Science and Technology Program, Taiwan International Graduate Program, Academia Sinica , Taipei 115, Taiwan.
authors.forename: Reta Birhanu
authors.initials: RB
authors.lastname: Kitata
authors.affiliation: Institute of Chemistry, Academia Sinica , Taipei 115, Taiwan.
authors.forename: Pei-Yi
authors.initials: PY
authors.lastname: Lin
authors.affiliation: Institute of Information Science, Academia Sinica , Taipei 115, Taiwan.
authors.forename: Wai-Kok
authors.initials: WK
authors.lastname: Choong
authors.affiliation: Institute of Chemistry, Academia Sinica , Taipei 115, Taiwan.|Department of Biochemical Sciences, National Taiwan University , Taipei 10617, Taiwan.
authors.forename: Tai-Du
authors.initials: TD
authors.lastname: Lin
authors.affiliation: Institute of Chemistry, Academia Sinica , Taipei 115, Taiwan.
authors.forename: Yi-Ting
authors.initials: YT
authors.lastname: Wang
authors.affiliation: Institute of Chemistry, Academia Sinica , Taipei 115, Taiwan.|Genome and Systems Biology Degree Program, National Taiwan University , Taipei 10617, Taiwan.
authors.forename: Shao-Hsing
authors.initials: SH
authors.lastname: Weng
authors.affiliation: Department of Internal Medicine, National Taiwan University Hospital , Taipei 10617, Taiwan.|National Taiwan University College of Medicine , Taipei 10051, Taiwan.|Institute of Biomedical Science, Academia Sinica , Taipei 115, Taiwan.
authors.forename: Pan-Chyr
authors.initials: PC
authors.lastname: Yang
authors.affiliation: Institute of Chemistry, University of the Philippines , Diliman Quezon City 1101, Philippines.
authors.forename: Susan D
authors.initials: SD
authors.lastname: Arco
authors.affiliation: Institute of Information Science, Academia Sinica , Taipei 115, Taiwan.
authors.forename: Ting-Yi
authors.initials: TY
authors.lastname: Sung
authors.affiliation: Institute of Chemistry, Academia Sinica , Taipei 115, Taiwan.|Department of Chemistry, National Tsing Hua University , Hsinchu 30013, Taiwan.|Molecular Science and Technology Program, Taiwan International Graduate Program, Academia Sinica , Taipei 115, Taiwan.
authors.forename: Yu-Ju
authors.initials: YJ
authors.lastname: Chen
mesh_terms: D045744:Cell Line, Tumor; D006801:Humans; D057230:Limit of Detection; D008175:Lung Neoplasms / Q000503:physiopathology; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008954:Models, Biological; D009154:Mutation; D040901:Proteomics / Q000379:methods*; D013997:Time Factors
chemical_list: D008565:Membrane Proteins