https://scholars.lib.ntu.edu.tw/handle/123456789/627082
標題: | Rapid High-pH Reverse Phase StageTip for Sensitive Small-Scale Membrane Proteomic Profiling | 作者: | Dimayacyac-Esleta, Baby Rorielyn T Tsai, Chia-Feng Kitata, Reta Birhanu Lin, Pei-Yi Choong, Wai-Kok Lin, Tai-Du Wang, Yi-Ting Weng, Shao-Hsing PAN-CHYR YANG Arco, Susan D Sung, Ting-Yi Chen, Yu-Ju |
關鍵字: | MASS-SPECTROMETRY; PEPTIDE IDENTIFICATION; FRACTION CONCATENATION; PROTEIN TOPOLOGY; LUNG-CANCER; CELL-LINES; CHROMATOGRAPHY; STRATEGY; SEPARATION; PREDICTION | 公開日期: | 15-十二月-2015 | 出版社: | AMER CHEMICAL SOC | 卷: | 87 | 期: | 24 | 起(迄)頁: | 12016 | 來源出版物: | Analytical chemistry | 摘要: | 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. |
URI: | https://scholars.lib.ntu.edu.tw/handle/123456789/627082 | ISSN: | 0003-2700 | DOI: | 10.1021/acs.analchem.5b03639 |
顯示於: | 醫學系 |
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