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Clinical Proteomics Mass Spectrometry Karolinska University Hospital, Solna Sweden Science for Life Laboratory, Stockholm
Filter Aided Sample Preparation (FASP) This protocol is used for buffer change and digestion of proteins before proteomics analysis by mass spectrometry. The filters are 10k and the maximum load is 250 μg of total protein. (See Wísniewski et al., Nature Methods, vol 6 no 5 May 2009). Materials: Urea (Sigma, U5128) HEPES pH 7.6 Dithiothreitol (DTT) Iodoacetamide (IAA) Trypsin (Promega, V511) MQ Note: Urea solutions must be prepared freshly and used within one day. Make your own HEPES buffer to avoid unknown components that interfere with the protein concentration measurements. 1M HEPES pH 7.6 can be kept as a stock, preferably in the freezer. Start by letting DTT and IAA (kept in the dark) reach room temperature, to avoid condensation in the containers. Note: It is easier to weigh the DTT and IAA in a weigh boat or an eppendorf tube (in this case it is best to dissolve the substance in small volume water before addition to the 10.0 ml volumetric flask). It is possible to make one large batch of 8M urea for all four urea buffers. Equipment Nanosep® Centrifugal Devices with Omega™ Membrane (Cat. Nr: OD010C34 10K, blue 100/pkg) or Microcon YM-10 (Millipore, Cat. No. number 42407) Refrigerated Bench-top centrifuge, temperature 20°C Heating block Reagent preparation: 50 ml 8 M urea buffer: - 24.0 g urea Dissolve the urea in ~40 ml MQ water. Add MQ water to reach a total volume of 50.0 ml Urea buffer 1 (UB1): 8 M urea buffer, 1 mM DTT, in 25 mM HEPES pH 7.6 - 250 µl 1 M HEPES pH 7.6 - 0,00154 g DTT Dissolve and mix the reagents in a 10.0 ml volumetric flask, dissolve in 8 M urea. Once all the reagents have dissolved fill up to 10.0 ml with 8 M urea. Urea buffer 2 (UB2): 8 M urea buffer, 25 mM IAA, in 25 mM HEPES pH 7.6 - 250 µl 1 M HEPES pH 7.6 - 0,04624 g Iodoacetamide Dissolve and mix the reagents in a 10.0 ml volumetric flask, dissolve in 8 M urea. Once all the reagents have dissolved fill up to 10.0 ml with 8 M urea.
Clinical Proteomics Mass Spectrometry Karolinska University Hospital, Solna Sweden Science for Life Laboratory, Stockholm
Urea buffer 3 (UB3): 8 M urea buffer, 25 mM HEPES pH 7.6 - 250 µl 1 M HEPES pH 7.6 Dissolve and mix the reagents in a 10.0 ml volumetric flask, dissolve in 8 M urea. Once all the reagents have dissolved fill up to 10.0 ml with 8 M urea. Urea buffer 4 (UB4): 0.25 M urea buffer, 50 mM HEPES pH 7.6, trypsin (enzyme to protein ratio 1:50). - 500 µl 1 M HEPES pH 7.6 - 312.5 µl 8 M urea Dissolve and mix the reagents in a 10.0 ml volumetric flask, dissolve in MQ water. Once all the reagents have dissolved fill up to 10.0 ml with MQ water. Note: The total volume of the buffers can be adjusted according to the number of samples. Methods Note: Make sure the proteins are completely dissolved before proceeding with the sample preparation. The samples should be a clear lysate. Note: If a lot of liquid remains in the spin filters, the centrifugation time needs to be increased. Different samples will behave differently, thus some samples will require longer centrifugation times. The recommended total amount of protein per sample is 200 µg per sample. Recommended sample concentration is 5-10 µg/µl. The collection tubes can hold ~400 µl, so they need to be emptied every second step. Day 1: 1. Determine protein concentration with Bio-rad DCC, unless this is done previously. 2. Add 200 µl MQ to wash the filter units and centrifuge them at 14,000xg for 15 min. (11.000 rpm on our centrifuge). 3. Add 200 µl UB1 to 30 µl sample and vortex, apply the sample-buffer mixture to the filter units and centrifuge at 14,000xg for 15 min. 4. Discard the flow-through. 5. Add 200 µl UB1 and centrifuge at 14,000xg for 15 min. 6. Add 200 µl UB2 and mix for 10 min in thermo-mixer at room temperature. 7. Centrifuge the filter units at 14,000xg for 15 min. 8. Discard the flow-through. 9. Add 200µl UB3 and centrifuge at 14,000xg for 15 min. 10. Repeat step 9. Make sure there is no remaining liquid on the filters. 11. Discard flow-through from the collection tube. 12. Add 100 μl UB4 with trypsin and mix at 37°C, 300 rpm, in thermo-mixer overnight.
Clinical Proteomics Mass Spectrometry Karolinska University Hospital, Solna Sweden Science for Life Laboratory, Stockholm
Day 2: 13. Centrifuge the filter units at 14,000xg for 15 min. 14. Add 50 µl MQ and centrifuge the filter units at 14,000 x g for 15 min. make sure there is no remaining liquid on top of the filters. 15. Transfer to a clean tube and measure the volume of the flow-through. Vortex and spin down before concentration measurement. 16. Determine peptide concentration with Bio-rad DCC.
Note: Peptide yield from the FASP methods depends on the sample and normally ranges from 60- 100%.
