AIM/HYPOTHESIS
After having tried several high-sensitivity methods to detect endogenous ORF2p in N2102Ep cells (LC-MS/MS, poly-HRP) without succes, Karin Wolters will perform a targeted MS assay on these samples. This means that the parameters of the MS will be set and optimized to detect peptides of ORF1p and ORF2p, based on a prior run with purified peptides of these proteins.
We have 3 samples, one essentially negative (HeLa), one positive (pLD401) and the endogenous cell line (N2102Ep). After this tester we will move to IPs on these cells (incl S-trap high-recovery protocol) to decomplex samples and elevate relative ORF protein levels.
MATERIALS
1) pLD401 | 100mg
2) N2102Ep | 100mg
3) HeLa S3 Flp-In | 100mg METHODS
Everything was performed with specific MS designated buffers.
1. Weigh out cell powder, equilibrate at RT for 30 secs, add extraction buffer to each tube (1:4 w/v), vortex to mix
2. Sonicate 2 x 5 x 2 sec at 4 (!!!) Amp – this setting at ERIBA ~equals to 2 Amp at RU.
Energy output sonication (J)
1) pLD401 | 13 + 13
2) N2102Ep | 16 + 17
3) HeLa S3 Flp-In | 16+153. Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R). Move cleared lysate to a clean 1.5ml tube.
4. Take 10ul from each sample to measure protein concentration using BCA assay. Put the rest of samples on ice for now.
Concentrations
1) pLD401 | 3.26 ug/ul
2) N2102Ep | 5.62 ug/ul
3) HeLa S3 Flp-In | 3.94 ug/ul5. Dilute each sample in 100 ul of 2 ug/ul final concentration. Take 50ul (100ug) per sample to continue S-trap protocol and save the rest for possible future WB analysis.
6. Prepare MS samples. S-trap micro column can handle <50-100ug of protein (http://www.protifi.com/wp-content/uploads/2018/08/S-Trap-micro-protocol-long.2.6.pdf).
7. dry down samples with speed-vac at RT (~30 min)
MS sample preparation was basically done according to the protocol in the link above. (NOT the high-recovery protocol).
8. Add 25ul of SDS solubilization buffer (5% SDS, 50mM TEAB (pH 7.55)) to the samples.
9. Reduce and alkylate disulfides.
10. Add 2.5ul 12% phosporic acid to the sample.
11. Place S-trap column(s) into a 2.0 mL vial (cut-off lid).
12. Add 165 μL of S-Trap binding/wash buffer (90% MeOH, 100 mM final TEAB, pH 7.1) into the S-Trap micro column. It will not flow through.
13. The next steps must be done as quickly as possible: Add 10 uL 100 ng/uL Trypsine stock solution to the sample and immediately mix by pipetting up and down, then immediately transfer the sample into the S-Trap binding buffer within the spin column. Again, mix by pipetting up and down.
14. Spin in bench-top centrifuge in a standard 2.0 mL tube at 4000 g/rcf until all solution has passed through (~30 sec). Remove flow through.
15. Wash by adding 150 μL S-Trap binding/buffer buffer to the spin column and centrifuging through (4000 g/rcf, 30 sec). Remove flow through. Repeat three times.
16. Place the spin column into a new 1.5 mL Lobind tube. *
17. Add 25 uL of Trypsin working solution to the top of the protein trap. The protein trapping matrix is highly hydrophilic and will absorb the solution. However, ensure there is no bubble atop the protein trap.
18. Cap the spin column loosely and incubate for 1 hr at 47 °C for trypsin. Most preferably use a water bath or thermomixer. DO NOT SHAKE. The cap MUST NOT form an air-tight seal.
19. Add 40 uL of 50 mM TEAB pH 8 to the protein trap. Centrifuge elution through at 4,000 g/rcf. *
20. Add 40 uL of 0.2% formic acid. Centrifuge elution through at 4,000 g/rcf. *
21. Elute hydrophobic peptides with 35 μL 50% acetonitrile, 0.2% formic acid (v:v). Centrifuge elution through at 4,000 g/rcf. *
* Elutions can be collected in same tube. If the elutions are collected separately, wash the tube with ACN before collecting the 50%ACN/0.2% formic acid elution.
Elutions were taken to the sixth floor, and stored frozen by Luciano.
RESULTS
DISCUSSION