Methods
Protocol Metabolite extraction
General metabolites extraction method (PDF)
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
Cell Lysis:
- 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
FASP method for protein Identification (PDF)
- 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
5 min
(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.