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A High-Efficiency Cellular Extraction System for Biological Proteomics

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Molecular Pharmacology & Chemistry Program, Sloan Kettering Institute, New York, New York 10065, United States
Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, United States
§ Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, New York 10065, United States
Weill Medical College of Cornell University, New York, New York 10065, United States
*E-mail: [email protected]. Tel: 646-888-3557.
Cite this: J. Proteome Res. 2015, 14, 8, 3403–3408
Publication Date (Web):July 8, 2015
https://doi.org/10.1021/acs.jproteome.5b00547
Copyright © 2015 American Chemical Society

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    Abstract

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    Recent developments in quantitative high-resolution mass spectrometry have led to significant improvements in the sensitivity and specificity of the biochemical analyses of cellular reactions, protein–protein interactions, and small-molecule-drug discovery. These approaches depend on cellular proteome extraction that preserves native protein activities. Here, we systematically analyzed mechanical methods of cell lysis and physical protein extraction to identify those that maximize the extraction of cellular proteins while minimizing their denaturation. Cells were mechanically disrupted using Potter–Elvehjem homogenization, probe- or adaptive-focused acoustic sonication, and were in the presence of various detergents, including polyoxyethylene ethers and esters, glycosides, and zwitterions. Using fluorescence spectroscopy, biochemical assays, and mass spectrometry proteomics, we identified the combination of adaptive focused acoustic (AFA) sonication in the presence of a binary poloxamer-based mixture of octyl-β-glucoside and Pluronic F-127 to maximize the depth and yield of the proteome extraction while maintaining native protein activity. This binary poloxamer extraction system allowed for native proteome extraction comparable in coverage to the proteomes extracted using denaturing SDS or guanidine-containing buffers, including the efficient extraction of all major cellular organelles. This high-efficiency cellular extraction system should prove useful for a variety of cell biochemical studies, including structural and functional proteomics.

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    Table 1: Protein extraction efficiency and activity of Jurkat-GFP cells lysed in 150 mM NaCl and 50 mM Tris–HC at pH 7.5, supplemented with 1% detergents and binary 1% detergents. Table 2: List of peptides identified in all proteomic analysis. Table 3: List of proteins identified in all proteomic analysis. Supp. Figure 1: Comparison of unique peptides identified using two-step versus one-step trypsin protein digestion. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.5b00547.

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