General Peptide Desalting Protocol with Buffers

Concentration and Purification of Peptides for LCMS Analysis on C18 Tip

NOTE: There are many routine methods for concentrating peptides using commercial products such as C18 tips that have been optimized for peptide desalting/concentration. Regardless of the particular method, we recommend that the method of choice be optimized for recovery and be amenable to peptide loading capacities of at least 10 µg input sample, for instance eluate from PTMScan® immunoaffinity purification (IAP).

A. Solutions and Reagents

NOTE: Prepare all solutions in containers that have never been autoclaved or exposed to soap, as detergents will interfere with LCMS analysis. All percentage specifications for solutions below are volume/volume.

1. StageTip Wetting Solution (50% Acetonitrile, 0.1% TFA) = 25 µL of Acetonitrile (Cell Signaling Technology, #40644), 20 µL of MS-grade Water (Cell Signaling Technology, #27732), 5 uL of 1% TFA (Cell Signaling Technology, #50573) for 50 uL final volume per assay.
2. StageTip Equilibration and Washing Solution (0.1% TFA) = 180 µL of MS-grade Water (Cell Signaling Technology, #27732), 20 uL of 1% TFA (Cell Signaling Technology, #50573) for 200 uL final volume per assay.
3. StageTip Elution Solution (40% Acetonitrile, 0.1% TFA) = 8 µL of Acetonitrile (Cell Signaling Technology, #40644), 10 µL of MS-grade Water (Cell Signaling Technology, #27732), 2 uL of 1% TFA (Cell Signaling Technology, #50573) for 20 uL final volume per assay.

   NOTE: Organic solvents are volatile. Tubes containing small volumes of these solutions should be prepared immediately before use and should remain capped as much as possible to prevent evaporation of organic components.

Equipment Not Included:

1. 1.5 mL microcentrifuge tubes
2. Centrifuge capable of handling 1.5 mL tubes
3. StageTip adaptors (Cell Signaling Technology, #58992)

B. Procedure

NOTE: All centrifugation steps in this section should be carried out at room temperature. Spin at a speed up to 2,000 x g, such that all of the solution passes through the tip in approximately 3 min. If necessary, spin for longer duration until entire solution passes through tip.

1. Cut the lid off of a 1.5 mL tube and place an adaptor on top. Place a C18 tip in the adaptor (Cell Signaling Technology, #58992). There should be enough room in the tube to collect ~100 µL of liquid without touching the C18 material at the bottom of the tip.
2. Equilibrate the C18 tip by pipetting 50 µL of StageTip Wetting Solution into the tip and centrifuging at 2,000 x g for approximately 3 min or until all of the solution has passed through. Transfer the C18 tip to a clean collection tube.
3. Add 50 µL of StageTip Equilibration and Washing Solution and centrifuge 3 min or until all of the solution has passed through. Repeat this step once for two total Equilibration & Washing steps. Discard flowthrough.
4. Load sample by centrifuging IAP eluate (approximately 100 µL initial total volume) through the C18 tip. Load IAP eluate 50 µL at a time for a total of two loading steps. Users may re-load the same 50 µL of eluate that passed through the C18 tip one additional time for a total of four loading steps. Peptides will be bound to C18 tip. Discard flowthrough after loading is complete. Confirm all of the solution has passed through after each loading step.
5. Wash the C18 tip by passing 50 µL of StageTip Equilibration and Washing Solution. Repeat this step once for two total Equilibration & Washing steps. Confirm all of the solution has passed through after each wash step. Discard flowthrough and transfer C18 tip to a clean collection tube.
6. Elute peptides off the C18 tip by centrifuging 10 µL of Stagetip Elution Solution. Repeat this step once for two total elutions. Confirm all of the solution has passed through after each elution step. Pool the two resulting eluates for a final volume of 20 µL.
7. Dry down the C18 tip eluate from the C18 tip purification in a vacuum concentrator (Speed-Vac) and store the dry peptides at -20°C until LCMS analysis will begin. At that time, re-dissolve the peptides in an appropriate solvent for LCMS analysis such as 5% acetonitrile, 0.1% TFA, 94.9% water (MS grade).