Mass spectrometry sample preparation

GuHCl_Trypsin PROTOCOL TO DIGEST COLLAGEN RICH TISSUES.

REAGENTS PREPARATION

0.4M Tris stock, pH 7.8

Add 2.42 g Tris (MW 121.14g) to a 100 ml beaker. Add 40 ml Milli-Q H₂O and dissolve. Adjust pH to 7.8 with 6M HCl.

200 mM IAA (I6125 SIGMA) in 0.1 M Tris buffer, pH 7.8. 

Add 0.0369 g IAA (MW 184.96) to a 1.5 ml tube. Add 750 ul Milli-Q H2O and 250 ul of 0.4M Tris stock and vortex. Store in the dark until ready to use. Prepare fresh. 

200 mM DTT  in 0.1 M Tris buffer, pH 7.8. 

Add 0.03 g DTT (MW 154.25) to a 1.5 ml tube. Add 750 ul Milli-Q H2O and 250 ul 0.4 M Tris stock and vortex. Store in ice until ready to use. 

4 M GuHCL buffer in 0.1 M Tris, pH 7.8

Add 38.2 g of Guanidine hydrochloride (GuHCl MW=95.53 g/mol) to 100 ml 0.1M Tris and vortex mix
 

1. Centrifuge samples (max, 2 mins) Remove liquid
2. Add 175 µL water (scale this and following volumes if does not cover)
3. Add 5 µL 200 mM DTT, heat 65 °C, 15 minutes. Cool to RT for approximately 10 min
4. Add 20 µL 200 mM IAA, 30 minutes @ RT
5. Add 50 µL 200 mM DTT to quench, 30 minutes @ RT
6. Centrifuge in table top centrifuge at 13,000 rpm for 2 min, room temperature.
7. Discard supernatant.
8. Add 50 µL 4M GuHCl buffer, vortex and sonicate for 10 minutes.
9. Incubate for 2h at 65 degrees.
10. Reduce the GuHCl concentration to a final concentration of < 0.5M by diluting the reaction mixture with 500 µL milliQ-H₂O and vortex.
11. Measure pH with pH strips
12. pH has to be approx. 8, so add 50 µL 0.4M Tris (Trizma base) if required.
13. Measure protein concentration with BCA assay.
14. Add Trypsin at a 25:1 protein: protease ratio (w/w), mix gently and incubate overnight (16h) at 37 °C.
15. Terminate digestion by adding TFA to 0.5-1 % final concentration. Remove particulate material by centrifuging at 14,000-16,000 x g for 10 minutes.
16. Desalt and Purify peptides on SOLA C18 solid phase extraction cartridges.
17. Dry in Speed vac.
18. Resuspend peptides in 20 ul loading buffer (1% ACN+0.5% TFA).