Filter Aided Sample Preparation (FASP) Method
The following protocol describes a method for generation of tryptic peptides from crude lysates for LC-MS analysis. The method allows analysis of detergent lysed cells and tissues. Therefore it is particularly suitable for studying entire proteomes and fractions containing biological membranes. Since the essential steps of the method occur within a filtration device the methods has been termed Filter Aided Sample Prep (FASP). The key features of the method making it superior over other sample preparation methods are:
1. The method provide protein digest that are free from nucleic acids and other cell components.
2. The method can be applied to samples containing high concentrations of detergents
3. There are no precipitation and the concentration of sample is kept high
4. In a single filter device 0.2-200 µg of total protein can be processed. Thus, FASP can be used upstream of separations of peptides such as OFFGEL and 2D- LC
5. The yield and purity of peptides can be monitored by UV-spectrometry allowing QC of the digest.
Solutions and Reagents
UA: 8 M urea (Sigma, U5128) in 0.1 M Tris/HCl pH 8.5. Prepare 1 ml per 1 sample.
IAA solution: 0.05 M iodoacetamide in UA. Prepare 0.1 ml per 1 sample.
Trypsin, Stock 0.4 µg/µl
0.5 M NaCl in water. Prepare 0.05 ml per 1 sample
ABC: 0.05M NH4HCO3 in water. Prepare 0.25 ml per 1 sample
Note: UA and IAA solutions must be freshly prepared and used within a day.
Microcon YM-30 (Millipore, Cat. MRCF0R030)
3M Empore HP Extraction disk cartridge (C18-SD); 7mm/ 3 ml (Varian Cat. No. 12144002)
Refrigenerated Bench-top centrifuge (Eppendorf 5415R), temperature 20°C
Multifuge 3L-R (Heraeus), (for peptide desalting in cartridges), temperature 20°C
Wet chamber with a rack for Eppendorf tubes
Thermo-mixer set to 20°C
2.1 Sample preparation
Lysis and thiol-reduction
There is no universal protocol to lyse any kind biological material but heating in the presence of high concentrations of SDS and reduction of disulfide bridges usually results in total or at least high extent of lysis.
Examples for total lysate preparation:
50 µl HeLa cell pellet (one ø 15 cm dish), 25 mg mouse liver or cerebellum are lysed by mixing with in 150 µl of solution containing 4% SDS, 100mM Tris/HCl pH 7.6, 0.1M DTT (lysis solution) and incubation at 95°C for 3 min. The DNA has to be sheared by sonication to reduce the viscosity of the sample. Before starting sample processing the lysate has to be clarified by centrifugation at 16,000 x g for 5 min.
1. The tissues have to be homogenized with a blender in the lysis solution before heating.
2. Avoid temperatures below 15°C and potassium salts to avoid precipitation of concentrated SDS.
3. 50 µl HeLa pellet or 25 mg brain or liver contains roughly 2 mg protein.
2.2 Sample processing
3. Mix up to 30µl of a protein extract with 200µl of UA in the filter unit and centrifuge at 14,000 x g for 15 min.
4. Add 200µlof UA to the filter unit and centrifuge at 14,000 x g for 15 min.
5. Discard the flow-through form the collection tube.
6. Add 100 µl IAA solution and mix at 600 rpm in a thermo-mixer for 1 min and incubate without mixing for 20 min.
7. Centrifuge the filter units at 14,000 x g for 10 min.
8. Add 100 µl of UA to the filter unit and centrifuge at 14,000 x g for 15 min. Repeat this step twice.
9. Add 100 µl of ABC to the filter unit and centrifuge at 14,000 x g for 10 min. Repeat this step twice.
10. Add 40 µl ABC with trypsin (enzyme to protein ratio 1:100) and mix at 600 rpm in thermo-mixer for 1 min.
11. Incubate the units in a wet chamber at 37°C for 4 -18 h.
12. Transfer the filter units to new collection tubes.
13. Centrifuge the filter units at 14,000 x g for 10 min.
14. Add 40 µl ABC and centrifuge the filter units at 14,000 x g for 10 min.
15. Acidify with CF3COOH and desalt the filtrate.
2.3 Yield determination
Concentration of the peptides can be estimated by UV spectrometer assuming that 0.1% solution of vertebrate proteins has at 280 nm an extinction of 1.1 absorbance units. (1mg/ml solution has 1.1 au). Always record a spectrum from 240-340 nm. It should have a distinct peak with a maximum at 270-280 nm and the extinction at 320 nm has to be 0.
1. Quarz cuvettes are required for correct absorption spectra.
2. Extinction values below 0.1 allow only rough protein estimation.