Last day of DNR extractions (hopefully)!

Grace and Kaitlyn helped me desalt the samples I speed vacuumed on Saturday, June 3. For the most part, I followed my protocol from December, adjusting the volume of final solvent needed at the end to accomodate for the 100 µg/100 µL protein samples I had.

Reagents required

Reagents were made by Jose using the following protocol on June 6.

• 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

Step 1: Label centrifuge tubes and columns

• For each sample to be desalted, label 2 centrifuge tubes and two macrospin column tubes
• Centrifuge tubes: L1 and L2 (ex. O04 L1 and O04 L2)
• Columns: A and B (ex. O04 A and O04 B)

Step 2: Reconstitute samples

• Added 100 µL Solvent B to each sample
• For sample O122, did the following
• Tested to see if sample is at pH 2
• If not, added 50 µL 10% formic acid
• Vortexed sample lightly to thoroughly mix solution
• Tested pH again
• Added additional 50 µL 10% formic acid
• Vortexed and tested pH
• Added final 50 µL of 10% formic acid (total formic acid added: 150 µL)
• Vortexed and tested pH. It was at pH 2
• Added 150 µL 10% formic acid to remaining samples

Jose told us that he needed 150 µL of 10% formic acid to get his samples to the desired pH. This was the same volume I used in December for my samples. We also needed 150 µL 10% formic acid per sample. Ideally, I would have tested the pH of all samples. However, we were out of pH strips, so I could only test 2.

Figure 1. pH test strips for 10% formic acid addition.

Step 3: 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

Step 4: 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

During this step, I spilled a small amount (a few specks) of salt from columns O04 A and O131 A. There also seemed to be a bubble in the column of O106 A.

Step 5: 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

Step 6: 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

Small specks of salt spilled from columns O14 A and O100 A. Additionally, Grace and I had a miscommunication and all columns associated with Round 2 samples were spun a total of four times instead of three. I don’t believe this will affect my peptide results, as they are washed out of the columns with Solvent A, not Solvent B.

Step 7: 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

Step 8: Evaporate peptides

• Using speed vacuum, evaporate samples to near dryness at 25ºC
• Speed vacuum start time: 4 p.m.

The original protocol states that this step should be done at 4ºC, but Emma said we could do it at 25ºC. This shortened our speed vacuuming time.

Table 1. Times samples were removed from the speed vacuum.

Time	Samples
4:49 p.m.	O01, O04, O06, O10, O14, O17, O21, O24, O26, O31, O35, O40, O43, O46, O49, O51, O56, O60, O64, O71, O101, O103, O173
4:57 p.m.	O12, O78, O113, O118, O124
5:08 p.m.	O08, O30, O32, O39, O52, O66, O90, O96, O100, O102, O106, O128, O140, O145, O147, OBLNK2
5:17 p.m.	O22
5:33 p.m.	O91, O99, O121, O122, O131

Step 9: Reconsitute peptides

• Added 100 µL of final solvent to each column
• Lighty vortexed all samples
• Centrifuged all samples down
• Stored samples in -80ºC freezer

For the most part, I’m all set for our July 10 mass spectrometry date!