TITLE: Peptide Desalting with a Vacuum Manifold
AUTHORS: Rebecca E. Hardman, Jeffrey Lewis, Tara Stuecker

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Desalting of peptides is an essential step for mass spectrometry workflows. We detail the steps for medium-throughput desalting (10-20 samples) with Oasis HLB Cartidges.</div></div>

[STEPS]
?. [Buffers]
Prepare buffers:        a. 0.1% TFA – Make 15 ml per sample plus 1-2 ml extra.              i. For example: if you are desalting 10 samples, you would make 152 ml (1  5 ml x 10 samples = 150 ml plus 2 ml              extra = 152 ml)        b. 70% Acetonitrile – Make 2 ml per sample plus 1-2 ml extra.
?. [Preparing Vacuum Manifold]
Set up vacuum manifold in chemical hood.    a. Attach feet to manifold.    b. Prepare one port for each sample you will be desalting as follows:            i. Remove yellow stoppers – store them in a conical tube to make sure they do not get lost. Stoppers must remain in any unused ports to maintain the vacuum.            ii. Insert stopcocks. Twist them to make sure they are secure, then align them all so they are facing out.            iii. Make sure fasteners on under side of stopcocks (bottom side of manifold) are attached firmly, then attach 18 gauge needles (still sheathed) to each.            iv. Assemble manifold by placing square waste bucket into glass stand. Place manifold onto glass stand. MAKE SURE YOU (CAREFULLY) REMOVE SHEATHS FROM NEEDLES WHEN YOU PLACE MANIFOLD ON GLASS COLLECTION STAND.            v. Connect hose from manifold to vacuum pump.             vi. Make sure all stopcocks are in the closed position (up/down).            vii. Place one Oasis desalting column on each stopcock and label the columns with each sample name.
?. [Preparing Columns]
Activate columns with 3ml 100% Acetonitrile.     a. Pipette Acetonitrile onto columns and let it flow through by gravity (do not turn on vacuum pump).
Be very careful not to let columns run dry during desalting process. For most steps, gravity will be enough for columns to drain. Only use the vacuum when necessary during the wash and elution steps. Watch the columns as they are draining. Once the liquid drains out, either immediately pipette the next step onto the column or close the stopcocks so it does not dry out.
?. [Preparing Columns]
Equilibrate column with 3 ml 0.1% TFA in water.    a. Pipette TFA onto columns and let it flow through by gravity (do not turn on vacuum pump). As soon as all liquid has passed into resin, close the stopcocks.
?. [Preparing Columns]
Remove plastic collection bucket from manifold and replace with collection rack containing 50 ml conical tubes for collecting flowthrough.     a. Label each conical tube with sample name.
These samples are collected in case the peptides do not bind to the column. In that case, they can be recovered from the flowthrough.
?. [Desalting]
Pipette 3 ml of each sample into desalting columns and let them flow through by gravity.     a. Repeat with another 3 ml until all of sample has applied through the column.
Be very careful not to mix up samples during this process and be careful not to let any columns run dry. If too many are ready at once, you can close the stopcocks.
?. [Desalting]
Wash columns with 3ml 0.1% TFA, collecting this first wash into the same 50 ml conical tubes that have the flowthrough. When all of wash solution has passed through column, close the stopcocks and wait until the wash step is complete for all samples
?. [Desalting]
Pull out collection rack and replace it with the plastic collection bucket for the remaining washes, which will not be collected. Put lids on flowthrough/wash tubes and freeze at -80°C.
?. [Desalting]
Repeat wash step 3 more times (3ml 0.1% TFA in water per wash) for a total of 4 washes.
Mark a line on each tube as you add each wash to keep track of how many washes you have done. Some columns will flow faster than others, making it essential to record the the wash steps for each sample.
If some columns are not draining by gravity, you can turn on the vacuum pump for this step. You generally do not need to dial up the vacuum--briefly turning on the pump is usually sufficient to restore flow. IF YOU DO USE THE VACUUM, MAKE SURE THE PRESSURE DOES NOT GO ABOVE THE MAX WRITTEN ON THE FRONT OF THE MANIFOLD.
?. [Desalting]
Wash with 3ml ultrapure water using gravity.
As with the previous wash, you can use the vacuum pump if necessary to restore flow.
?. [Desalting]
Remove waste container from glass base and replace it with your elution tubes (15 ml conical tubes in collection rack—make sure you label your tubes with your sample names both on the sides of the tubes and the lids.)
?. [Desalting]
Elute with 3 ml 70% Acetonitrile    a. Use gravity until there is no more visible liquid, and then pull a vacuum to get out the last remaining liquid.
?. [Desalting]
Remove samples from collection rack and put lids on tubes.    a. If lyopholizing, freeze samples in liquid nitrogen.
?. [Cleaning Manifold]
To clean the vacuum manifold:    a. Dispose of acetontrile and TFA waste according to institutional guidelines.    b. Carefully remove and discard needles in sharps box.    c. Soak stopcocks in beaker with detergent to clean, then rinse well.    d. Rinse manifold with water, squirt soap solution through all ports and rinse thoroughly with purified water.    e. Do a final rinse with ethanol and dry on bench.