11/30/2016

Desalting

For this procedure you will need:

1) Solvent A = 60% acetonitrile + 0.1% trifluoroacetic acid

2) Solvent B = 5% acetonitrile + 0.1% trifluoroacetic acid

3) Final Solvent = 3% acetonitrile + 0.1% formic acid

4) Macrospin columns (Sample capacity: 0.03-300ug, Elution volume 50-150ul, Bed volume 300ul)

Procedure

1) Reconstituted samples by adding 100ul of Solvent B. Adjusted pH to less than 2 using 10ul of 10% formic acid at a time (I ended up using 80ul of 10% formic acid). I just tested the first sample and added the same amount of formic acid to the second one. I checked the pH to ensure it was also less than 2. Then I added 80ul of 10% formic acid to the remaining samples.

2) Realized none of the centrifuge in our lab fit all 22 tubes when you have the columns in them. The columns make them wider so I had to use two different centrifuges. Prepared macrospin columns by adding 200ul Solvent A to each column. Spun for 2000 rpm for 3 minutes (repeat 3 times for a total of 4 times). Discarded remaining liquid at the bottom of tube everyother addition to accomodate room for next round.

3) Equilibrated columns by adding 200ul Solvent B to each column. Spun for 2000 rpm for 3 minutes (repeat 2 times for a total of 3 times). Discarded remaining liquid at the bottom everyother time to accomodate room for next round.

4) Loaded protein onto column by pipetting all of the sample onto column. Spun at 3000rpm for three minutes. Collected the liquid at the bottom of the tube and put back on the column for a second round. Peptides are now in the columns. Transferred this remaining liquid to a new labeled tube (just in case).

5) Wash salts through column by adding 200ul Solvent B and spinning at 3000 rpm for three minutes (repeat twice for a total of 3 times). Saved remaining liquid at the bottom of each tube by transferring it to a new labeled tube (just in case).

6) Transfered columns to clean collection tubes. Added 100ul Solvent A, spun at 300 rpm for 3 minutes (repeated once for a total of 2 times). This liquid contains your peptides.

7) Evaporated samples to near dryness at 4C (took about 4 hrs).

8) Reconstituted peptides in 100ul 3% acetonitrile + 0.1% formic acid. Placed samples in -80C.