Protocol Metabolite extraction
Keep samples on ice or cool as possible especially during vortex
Perform all steps in epi-tube (2ml) (30 min)
- Resuspend cell pellet in (50mM NaCl) and transfer to epi-tube (2ml) (Ice)
- Spin at 10,000xg for 15 min (-9C)(15 min)
- Remove PBS supernatant
- Extraction ( 2X[100%H2O]:[WET CELL PELLET]) (5min/sample)
- Resuspend cell pellet in 3X 100% H2O
- Vortex till homogenous murky mixture (use powerful vortexer)
- Sonicate 60% duty cycle power 5 for (5min/sample) (keep cold!)
- Add equal volume of 100% MeOH
- Vortex 30s Spin at 20,000xg for 15 min (-9C)
- Pool Top MeOH /H2O into [Metabolite Extract]
- Wash ( [50%MeOH]:[Cell Debris]) (30 min)
- Add [50% MeOH] to [Cell Debris]
- Vortex 30s (use powerful vortexer)
- Spin at 20,000xg for 30 min (-9C)
- Pool 50% MeOH into [Metabolite Extract]
Acetone Precipitation: (2.5 hours)
- (5:1 v/v) Add 5x volume of -80C Acetone to [Metabolite Extract]
- Freeze in -80C for two hours (minimum)
- Spin at 20,000xg for 5 min
- Remove Supernatant and transfer to fresh tube
Concentration: (2.5 hours)
- Dry in speed vac
- Resuspend in 50% MeOH
Filter –Aided Sample Preparation for nano-LCMS analysis
- Rinse Nanosep molecular weight cut-off spin filter (3K) with 200 uL of 50 mM Amm.
Bicar. pH 7.5 and
centrifuge at 5K rpm for 5 min (2x).
- After collecting the purified proteins (beads: Pull-downs, SEC fraction …) place the
material onto the
spin filter and exchange the buffer using the buffer mentioned in the first step.
- CAUTION: it is important to eliminate incompatible detergents like Triton, Tween,
SDS, and PEG. It
is recommended to perform the digestion in Amm. Bicar. pH 7.5.
- CAUTION: it is important to eliminate incompatible detergents like Triton, Tween, SDS, and PEG. It
- Repeat the second step multiple times until all incompatible detergents/salts are exchanged.
- Add 10 uL of 100 mM DTT solution to your sample and incubate at RT for 30 min.
- Wash the sample by adding the 200 uL of Amm. Bicarb. and centrifuge at 5K rpm for
(centrifugation time may increase up to 15 min depending on the sample. Centrifugation speed may
increase to 8K rpm).
- Add 10 uL 100 mM IAM solution to the sample and incubate in the dark at RT for 20 min.
- Repeat step number five.
- Add sequencing grade trypsin at a ratio of 1:50 trypsin to total protein after adjusting
to volume to 50 uL
of Amm. Bicarb. and incubate at 37C for 8-16 hours.
- Spin the sample on the next day to collect the tryptic peptides (volume should be around 50 uL.
- SpeedVac the collected peptides to concentrate the sample for 20 min.
- Vortex the sample for 1 min.
- Centrifuge the sample at 15 K for 10 min.
- Transfer only 20 uL of the sample to mass spec sample vial and submit it to the mass spec facility.