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We use Millipore ZipTips, micro-C18, part no. ZTC18M096; and Hamilton syringes, 1701N (10 μL) and 1710N (100 μL) (or 1701RNR and 1710RNR) (blunt needles, as used in HPLC). Note: The manufacturer of the ZipTips suggests using a pipetter, which works, but peptide yield is 2-3x better if you use a syringe.
You will need the following solutions:
1) 0.1 % TFA in good quality water
2) 50/50 mixture of solution 1 and acetonitrile (50/50).
Procedure:
Prepare the sample: Extracts from the gel digest will contain about 50-100 μL of ~50% acetonitrile (ACN). Reduce the volume to about 5 μL in a vacuum centrifuge (Speedvac). This concentrates the sample but also reduces the ACN concentration to <5%. It is better not to allow the samples to go to dryness because sample loss will occur, but if a sample goes dry, reconstitute with 5 μL of 50/50. (Samples can be stored at this point at +4, -20 or -80°C. (ACN and water separate into two phases at -20°C, but for these small samples, it doesn’t seem to matter.)
Condition the tip: Rinse the tip with 50/50, and then equilibrate with 0.1% TFA. We prefer to do a single 100 μL rinse with each of the two solutions, using a 100 μL syringe. At the very start of the 50/50 wash, tap the syringe plunger and squirt through a bit of solvent to get rid of air bubbles.
Adsorb: Dilute the sample with 90 μL of 0.1% TFA. This is to reduce the concentration of ACN so that the peptides will bind to the C18 resin in the ZipTips. Attach the conditioned ZipTip to a 100 μL syringe (just press on) and draw the sample solution back and forth through the ZipTip at least ten times. Do this rather slowly to allow adsorption to take place. Keep the tip wet: don’t allow air bubbles to get trapped in the tip.
Rinse: Empty a syringe with ~100 μL of 0.1% TFA slowly through the tip to rinse out salts and other water-soluble impurities.
Elute: There are two ways to do this.
1. Put 3 μL of 50/50 into a 200 μL PCR tube. With a 10 μL pipetter or syringe, draw the solution slowly back and forth through the C-18 resin at least 5-10 times to improve
recovery.
2. Put 4 μL of 50/50 in a 10 μL syringe. Put the tip on the syringe and elute slowly. Discard the first one μL, then collect the rest. Do the elution slowly to allow
desorption to take place.
Store eluted samples at -20 or -80°C. They should be analyzed as soon as possible after elution, because peptides tend to irreversibly adsorb to the tube walls or otherwise
vanish.