Not Salty about Desalting

Our final day of protein extractions! :tada: After a late night (or early morning) using the speed vacuum yesterday, I am not at all sad, salty or mad about wrapping this baby up :joy:

Desalting allows us to isolate the peptides in our samples and prepare them for Mass Spectrometry.

Materials required:

Reagents were made by Rhonda using the following protocol.

• Solvent A = 60% acetonitrile + 0.1% trifluoroacetic acid (300ul/sample)
• Solvent B = 5% acetonitrile + 0.1% trifluoroacetic acid (500ul/sample)
• Final Solvent = 3% acetonitrile + 0.1% formic acid (100ul/sample)
• 10% formic acid
• Macrospin columns (Sample capacity: 0.03-300ug, Elution volume 50-150ul, Bed volume 300ul)
• 2 sets of 11 each, labelled
• Snaptop centrifuge tubes
• 2 sets of 11 each, labelled

Sample reconstitution:

• Added 100 µL Solvent B to each sample
• For 1-2 samples, did the following
• Tested to see if sample is at pH 2
• If not, added 10-20 µL 10% formic acid
• Vortexed sample lightly to thoroughly mix solution
• Tested pH again
• Kept adding 10% formic acid in 10 µL increments until sample is at pH 2
• Added 180 µL of 10% formic acid to sample O124
• Based on the amount of formic acid to the 1-2 test samples, added formic acid to the rest of the samples
• Added 100 µL of 10% formic acid to sample O55 and tested pH
• Added an extra 50 µL of 10% formic acid to O55 and tested pH
• pH was at 2 units
• Decided to add only 150 µL 10% formic acid to remaining samples
• Checked pH of all samples

Figure 1. pH test strips for all samples. With the exception of O124, I added 150 µL of 10% formic acid to each sample. I added 180 µL of 10% formic acid to O124. Note: “O124” should read “O127”

Wash columns:

• Took 1 set of 11 Macropsin columns
• Removed caps from top and bottom of columns
• Placed columns in set of collection tubes labelled “A”
• Added 200 µL of Solvent A to each column
• Spun columns for 3 minutes at 2000 rpm
• Repeated 3 more times for a total for 4 spins
• Discarded remaining liquid in column every other spin to accomodate additional liquid

Equilibrate columns:

• Added 200 µL of Solvent B to each column
• Spun columns for 3 minutes at 2000 rpm
• • Repeated 2 more times for a total of 3 spins
• Discarded remaining liquid in column after the second and third spin to accomodate additional liquid

Load protein on columns:

• Vortexed samples with protein digest once more to thoroughly mix solution
• Added 30 µg of protein digest to each column
• Pipetted total volume of liquid in sample snaptop centrifuge tube into the associated column
• ex. “O07 MT” (snaptop centrifuge tube with protein digest) –> “O07 A” (collection tube with column)
• Spun columns for 3 minutes at 3000 rpm
• Pipetted liquid that flowed through column
• Put flow-through back on column
• Spun columns again for 3 minutes at 3000 rpm
• Peptides are now in the columns
• Transfered remaining liquid to the first set of previously labelled snaptop centrifuge tubes
• ex. “O07 A” (collection tube with column) –> “O07 L1” (snaptop centrifuge tube for liquid flow-through)
• Store snaptop centrifuge tubes in -80ºC freezer

Wash salts through columns:

• Added 200 µL of Solvent B to each column
• Spun columns for 3 minutes at 3000 rpm
• Repeated 2 more times for a total of 3 spins
• Transfered remaining liquid to the second set of previously labelled snaptop centrifuge tubes
• ex. “O07 A” (collection tube with column) –> “O07 L2” (snaptop centrifuge tube for liquid flow-through)
• Stored snaptop centrifuge tubes in -80ºC freezer

Elute peptides:

• Transfered column contents to the second set of previously labelled Macrospin columns
• ex. “O07 A” (collection tube with column) –> “O07 B” (new collection tube with column)
• Added 100 µL of Solvent A to each column
• Spun columns for 3 minutes at 3000 rpm
• Repeated 1 more time for a total of 2 spins
• Peptides are now in the liquid
• Disposed of columns and keep collection tubes with liquid

Evaporate peptides:

• Using speed vacuum, evaporate samples to near dryness at 4ºC
• Because someone else was using the speed vacuum when we arrived at the Genome Sciences building, we compromised and ran our samples with theirs at 8ºC.
• Speed vacuum start time: 4 p.m.

Table 1. Times samples were removed from speed vacuum. Samples were loaded at 4 p.m. at a temperature of 8ºC with refrigeration.

Sample	Time Removed from Speed Vacuum
O07	6:31 p.m.
O15	6:20 p.m.
O37	6:20 p.m.
O47	6:20 p.m.
O55	6:20 p.m.
O77	5:55 p.m.
O107	5:55 p.m.
O119	6:20 p.m.
O127	6:20 p.m.
O142	6:20 p.m.
OBLANK	5:55 p.m.

Reconsitute peptides:

• Added 60 µL of final solvent to each column
• Lighty vortexed all samples
• Centrifuged all samples down
• Stored samples in -80ºC freezer

We’re done with extractions! We’ll load these samples on the Mass Spectrometer in late January.