A High-Efficiency Cellular Extraction System for Biological Proteomics

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.