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 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 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 binary poloxamer-based mixture of octyl-β-glucoside and Pluronic F-127 to maximize the depth and yield of proteome extraction while maintaining native protein activity. This binary poloxamer extraction system allowed native proteome extraction, comparable in coverage to proteomes extracted using denaturing SDS or guanidine containing buffers, including 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.

Automated sample preparation for high-throughput single-cell proteomics

“…1h). The number of proteins identified and quantified using urea lysis is comparable to that from similar label-free studies 4 . Almost all of these proteins were identified and quantified by mPOP as well (2,438 proteins), but mPOP samples contained an additional 953 proteins, Fig.…”

“…Methods for preparing sub-microgram protein samples for LC-MS/MS often use sophisticated custom-made equipment [1][2][3] and generally lyse cells by detergents or chaotropic agents like urea 3,4 .…”

“…Techniques using these chemicals are robust but require that the chaotropic agents or detergents be removed before MS analysis since these chemicals are incompatible with MS 4 . Some cleanup methods, such as SP3 5 and iST 6 perform very well even for microgram samples 4,7 . Yet losses are more significant for the preparation of low-abundance samples, such as single cells.…”

“…Thus, avoiding cleanup stages can reduce losses while increasing throughput and consistency 8 . A cell lysis method that does not require MS-incompatible chemicals and thus can be used for LC-MS/MS without cleaning is focused acoustic sonication (FAS) 4,9 . We successfully used FAS to obviate cleaning up single-cell lysates and to develop Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS) 10 .…”

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

“…Many different cell lysis techniques exist, including mechanical/physical approaches employing crushing impact, shearing, and/or pressure, as well as chemical/ enzymatic approaches, each with different pros and cons 49,50 . Each investigator is encouraged to explore methods most suitable for their analyses, keeping in mind that any chosen approach for cell breakage and protein extraction is likely to introduce biases 51,52 necessitating empirical optimization (discussed below). Mechanical methods may produce high heat and/or shearing forces which can disrupt protein complexes.…”