Mass Spectrometry Appropriate Protocol for Cell Lysis using Probe Sonication

  1. Wash cell pellet 3x with 10 volumes of 50 mM Ammonium Bicarbonate. Pellet cells gently between washes by centrifuging at ~5000 rcf for 3 minutes. Carefully pipette supernatant off after final wash, so as to not disturb the cells.
  2. Add 5 volumes of lysis buffer, and vortex to suspend. The minimum volume for probe sonication is 100 uL in a 0.5 mL eppendorf tube. Standard lysis buffer for proteomics applications is 0.1%‐0.25% w/v Rapigest SF ( in 50 mM Ammonium Bicarbonate. To increase coverage for membrane‐bound and integral membrane proteins, substitute with 0.5% w/v PPS Silent Surfactant ( in 50 mM Ammonium Bicarbonate.
  3. Place samples on ice.
  4. Perform probe sonication with 3 cycles of 15 seconds on, 5 seconds off, at 20% power. If possible, keep samples on ice during sonication to prevent excessive heating.
  5. Centrifuge samples at 15,000 rcf for 5 minutes, and measure protein concentration of the supernatant using a mini Bradford (‐ or micro BCA ( assay, depending upon the lysis buffer composition.