title: A High-Efficiency Cellular Extraction System for Biological Proteomics.
abstract: 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.
authors.forename: Avantika
authors.initials: A
authors.lastname: Dhabaria
authors.forename: Paolo
authors.initials: P
authors.lastname: Cifani
authors.forename: Casie
authors.initials: C
authors.lastname: Reed
authors.affiliation: ‡Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115, United States.
authors.forename: Hanno
authors.initials: H
authors.lastname: Steen
authors.affiliation: §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.
authors.forename: Alex
authors.initials: A
authors.lastname: Kentsis
mesh_terms: D002457:Cell Extracts / Q000737:chemistry* / Q000302:isolation & purification; D002460:Cell Line; D045744:Cell Line, Tumor; D005591:Chemical Fractionation / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D003902:Detergents / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D010455:Peptides / Q000378:metabolism*; D020442:Poloxamer / Q000737:chemistry; D020543:Proteome / Q000302:isolation & purification / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D013010:Sonication / Q000379:methods; D013050:Spectrometry, Fluorescence; D013347:Subcellular Fractions / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D002457:Cell Extracts; D003902:Detergents; D010455:Peptides; D020543:Proteome; D020442:Poloxamer
keywords: adaptive focused acoustics; binary detergent; cell lysis; protein extraction