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Materials (Basel). 2016 May 18;9(5). pii: E385. doi: 10.3390/ma9050385.

Streamlined Membrane Proteome Preparation for Shotgun Proteomics Analysis with Triton X-100 Cloud Point Extraction and Nanodiamond Solid Phase Extraction.

Author information

1
Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 10617, Taiwan. minhphd@gmail.com.
2
Institute of Biotechnology, Vietnam Academy of Science & Technology, Hanoi 10600, Vietnam. minhphd@gmail.com.
3
Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 10617, Taiwan. amy3_wen@hotmail.com.
4
Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 10617, Taiwan. hli@gate.sinica.edu.tw.
5
Agricultural Biotechnology Research Center, Academia Sinica, Taipei 11529, Taiwan. pation35@gmail.com.
6
Agricultural Biotechnology Research Center, Academia Sinica, Taipei 11529, Taiwan. yetran@gate.sinica.edu.tw.
7
Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 10617, Taiwan. hchang@gate.sinica.edu.tw.
8
Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 10617, Taiwan. cchan@po.iams.sinica.edu.tw.

Abstract

While mass spectrometry (MS) plays a key role in proteomics research, characterization of membrane proteins (MP) by MS has been a challenging task because of the presence of a host of interfering chemicals in the hydrophobic protein extraction process, and the low protease digestion efficiency. We report a sample preparation protocol, two-phase separation with Triton X-100, induced by NaCl, with coomassie blue added for visualizing the detergent-rich phase, which streamlines MP preparation for SDS-PAGE analysis of intact MP and shot-gun proteomic analyses. MP solubilized in the detergent-rich milieu were then sequentially extracted and fractionated by surface-oxidized nanodiamond (ND) at three pHs. The high MP affinity of ND enabled extensive washes for removal of salts, detergents, lipids, and other impurities to ensure uncompromised ensuing purposes, notably enhanced proteolytic digestion and down-stream mass spectrometric (MS) analyses. Starting with a typical membranous cellular lysate fraction harvested with centrifugation/ultracentrifugation, MP purities of 70%, based on number (not weight) of proteins identified by MS, was achieved; the weight-based purity can be expected to be much higher.

KEYWORDS:

cloud point extraction; diamond-enhanced proteolytic digestion; mass spectrometry; membrane proteomics; nanodiamond; salt-induced phase separation

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