title: Dissolving capability difference based sequential extraction: A versatile tool for in-depth membrane proteome analysis.
abstract: Profiling membrane proteins would facilitate revealing disease mechanism and discovering new drug targets as they play essential roles in cellular signaling, substrate transport, and cell adhesion. However, the analysis of membrane proteins still remains a challenge due to their high hydrophobicity, as well as the suppression effect of high abundant soluble proteins. In this work, to achieve a membrane proteome profiling, a sample preparation strategy based on sequential extraction at the protein level assisted by a range of extraction reagents with different dissolving capabilities, followed by nano-RPLC-ESI-MS/MS analysis was developed and applied for HeLa cell line analysis. It was found that with progressively harsher extraction reagents (i.e., 2 M NaCl, 4 M urea, 0.1 M Na2CO3, and 10% 1-dodecyl-3- methyl-imidazolium chloride (C12ImCl) performed, much more high hydrophobic proteins and low abundant proteins were identified. With our developed strategy, 5553 of the identified proteins (4419 gene products) were annotated to be membrane proteins and 2573 proteins (2183 gene products) have at least one transmembrane domain, to our best knowledge, which is the most comprehensive membrane proteome dataset for HeLa cell line. Notably, 110 of the identified membrane proteins were discovered in the "missing proteins" list referred to those in the neXtProt database. All above results indicated that our strategy has great potential to tackle the difficult but relevant task of identifying and profiling membrane proteins.
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, National Chromatographic Research and Analysis Center, 457 Zhongshan Road, Dalian 116023, China; Graduate School of Chinese Academy of Sciences, Beijing 100039, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Fang
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, National Chromatographic Research and Analysis Center, 457 Zhongshan Road, Dalian 116023, China.
authors.forename: Qun
authors.initials: Q
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, National Chromatographic Research and Analysis Center, 457 Zhongshan Road, Dalian 116023, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Li
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, National Chromatographic Research and Analysis Center, 457 Zhongshan Road, Dalian 116023, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Liang
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, National Chromatographic Research and Analysis Center, 457 Zhongshan Road, Dalian 116023, China. Electronic address: lihuazhang@dicp.ac.cn.
authors.forename: Lihua
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, National Chromatographic Research and Analysis Center, 457 Zhongshan Road, Dalian 116023, China.
authors.forename: Yukui
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D056148:Chromatography, Reverse-Phase; D030562:Databases, Protein; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D020543:Proteome; D012995:Solubility; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D008565:Membrane Proteins; D020543:Proteome
keywords: Membrane proteome profiling; Missing proteins; Sample preparation; Sequential extraction