The tough exterior cuticle of Caenorhabditis elegans makes the nematode resilient to lysis and impedes proteomic analyses, particularly when preservation of the native conformation and biological activity of proteins prohibits the use of chaotropes or detergents. Total disruption of nematodes resulting in maximal protein yields from mild, non-denaturing buffers were obtained using the PCT Shredder device followed by pressure cycling technology (PCT).Materials
Barocycler (Cat# NEP-3229; Pressure BioSciences)
PULSE Tubes (Cat# FT00500-S; Pressure BioSciences)
PCT Shredder Kit (Cat# KSFT-00001; Pressure BioSciences)
CE PrEP Kit (Cat# KPCE-01; Pressure BioSciences)
Ultrafree-CL Centrifugal Filtration Devices (Cat# UFC40SV; Millipore, Billerica, MA, USA)Methods Nematode Cultures
Mixed C. elegans populations (larval through adult hermaphroditic stages) were collected from cultures and the biomass was pelleted centrifugally. The pellet was extensively washed to remove residual Escherichia coli and the live nematodes were concentrated in an Ultrafree-CL centrifugal filtration device.Pressure Cycling Technology (PCT)
Fifty milligrams of the resulting live worm pellet were mixed with 100 mg SiC abrasive from the CE Pressure Enhanced Processing (PrEP) Kit and flash frozen directly in PULSE Tubes (specialized containers for processing samples at high pressure). The frozen sample was rotationally ground with the movable PCT Shredder ram until the entire sample was expressed through the perforations of the stationary disc in the PULSE Tube. 1500 microliters of 50 mM K3PO4 pH7.2 containing protease inhibitors were added and PCT was performed for 40 cycles at 35,000 psi. Subsequently, the PULSE Tubes were evacuated and nematode debris pelleted centrifugally at 10,000×g for 10 min. The supernatants were reserved for protein assay and the pellets were examined microscopically following Trypan Blue staining.Results
The resiliency of C. elegans is exemplified by the recovery of live nematodes from the Space Shuttle Columbia crash debris (1). In physiological buffers, nematodes can withstand pressure to 20,000 psi with a 2.3% survival rate. Adult hermaphrodites are selectively destroyed at lower pressures, while larvae are more resilient. All nematodes were eradicated at 35,000 psi, but protein yields remained low and only 10–14% of cuticles were Trypan Blue permeable. Under these conditions, some viable embryos remain. PCT has previously been described for the synchronization of C. elegans cultures (2).
The PCT Shredder disrupted all nematodes including dauer stage larvae. Protein yields in physiological buffers were six times greater with the PCT Shredder than with high pressure alone, and an order of magnitude higher when the PCT Shredder was used in combination with freezing and abrasives. Protein yields were further doubled when chaotropic reagents supplied with the CE PrEP kit were used.
By comparison, temperature fluctuations during thermostated bead beating resulted in highly variable protein recoveries. Furthermore, large protein aggregates observed microscopically in bead beating preparations were not observed in PCT Shredder preparations. Moreover, protein denaturation was observed in bead beating preparations, resulting in the gradual loss of soluble protein with each successive cycle.
Address correspondence to Pressure BioSciences, Inc., 14 Norfolk Avenue, South Easton, MA 02375, (508) 230-1828, email@example.com, www.pressurebiosciences.com.
Click here for more information