Jan 23, 2022
Version 1
General proteomics FASP (Filter-Aided Sample Preparation) V.1
- Jacob Waldbauer1,
- Lichun Zhang1
- 1University of Chicago
Protocol Citation: Jacob Waldbauer, Lichun Zhang 2022. General proteomics FASP (Filter-Aided Sample Preparation). protocols.io https://dx.doi.org/10.17504/protocols.io.if7cbrn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: June 14, 2017
Last Modified: January 23, 2022
Protocol Integer ID: 6367
Abstract
Spin-filter based protein digestion and purification for bottom-up proteomics
Guidelines
It is important that nearly all liquid passes through spin filters after each centrifugation step. If a substantial amount of liquid (more than 75 μL) remains on filter after 10 minutes of centrifugation, spin for longer.
Materials
MATERIALS
Pierce trypsin ProteaseThermo Scientific
Tween 20Bio-Rad LaboratoriesCatalog #170-6606-MSDS
Optima LC/MS grade WaterFisher ScientificCatalog #W6-4
UreaFisher ScientificCatalog #U15-500
Ammonium bicarbonateFisher ScientificCatalog #A643-500
Deoxycholic AcidbioworldCatalog #40430004-1
Optima LC/MS grade AcetonitrileFisher ScientificCatalog #A955-212
Formic Acid (Optima LC/MS)Fisher ScientificCatalog #A117-50
Ethyl AcetateFisher ScientificCatalog #E145-4
Before start
Passivate Vivacon filter unit (Satorius Vivicon 500, 30,000 MWCO) and collection tube overnight in 5% (v/v) Tween-20 in MilliQ water. The next day, rinse with nanopure water until no suds, then rinse 3x with LC-MS water.
Day 1
Day 1
Prepare fresh buffers in LC-MS water:
Exchange buffer: 8M urea, 0.2% (w/v) deoxycholate, 1M ammonium bicarbonate (pH 8)
Digestion buffer: 0.2% (w/v) deoxycholate, 50 mM ammonium bicarbonate (pH 8)
Trypsin dissolution/peptide recovery buffer: 50 mM ammonium bicarbonate (pH 8)
Dispense 25-50μL (depending on concentration) reduced, alkylated protein extract to filter unit.
Fill filter units with 200 μL exchange buffer, mix with pipet.
Spin at 14,000g for 10 min, discard filtrate.
Repeat steps 3 and 4 three more times.
Wash filter unit with 200 μL digestion buffer, and spin at 14,000g for 10 min. Do this a total of 2 times.
Transfer filter unit to passivated collection tube.
Add 110 μL digestion buffer and desired amount of trypsin (~1:50 trypsin/protein), dissolved in 50 mM ammonium bicarbonate, keeping trypsin on ice or at 4° C. Incubate at 37° C overnight. Parafilming tubes is recommended to prevent evaporation.
Day 2
Day 2
Remove parafilm if applicable and centrifuge tubes at 14,000g for 10 min. Do not discard filtrate!
Add 50 μL of peptide recovery buffer to filters, spin at 14,000g for 10 min. Do this step a total of 2 times.
Transfer the filtrate to a LoBind tube.
Add 900μL ethyl acetate and 2.5 μL TFA to filtrate, vortex. Precipitate may be visible.
Sonicate in a bath sonicator for 10s, and centrifuge at 16,000g for 10 min.
Carefully remove upper organic layer with a pipet without disturbing phase boundary.
Repeat steps 12, 13 and 14 two more times (Note: Only add 900μL ethyl acetate, no more TFA)
Place sample uncovered in Thermomixer and dry at 60° C for 5-10 minutes until ethyl acetate is gone.
Freeze sample solid at -80° C, Centrivap to dryness.