laboratory
Desalting strategy for protein content < 20 μg

StageTips C18 desalting

What is it for?

Any contaminating substances like detergents, salts, nucleic acids etc. impair efficient ionization of peptides and decrease the depth of mass spectrometry analysis. Non-purified samples can also lead to a physical damage of the MS instruments.

Original citation can be found here.

Solvents

  • C18 Macherey-Nagel discs
  • Buffer A (equilibration buffer) - 0.1% formic acid in Milli-Q water
  • Buffer B (elution buffer) - 50% Acetonitrile, 0.1% formic acid
  • Buffer C (wash buffer) - 5% Acetonitrile, 0.1% formic acid
  • 100% Acetonitrile

Sample preparation:

  1. Stack 2 layers of C18 material in a 200 μl pipette tip (prepare one for each sample!)

  2. Equilibrate the StageTips:

    1. Add 200 μL methanol to the StageTip —> centrifugation at 2.000 rpm for 1 min
    2. Add 100 μL buffer B to the StageTip —> centrifugation at 2.000 rpm for 1 min
    3. Add 100 μL buffer A to the StageTip —> centrifugation at 2.000 rpm for 1 min
    4. Add 100 μL buffer A to the StageTip —> centrifugation at 2.000 rpm for 20 sec
    5. Make sure to keep a little bit (approximately 1-2 mm) of buffer A on top of the C18-material.

  3. Load the samples (acidified, volume approx. 100 μl) onto the StageTips and centrifuge at 2.000 rpm for 3 min (adjust the time of centrifugation, if necessary)
    Note: if sample volume exceeds 100 μl repeat the steps

  4. Wash the StageTips with 100 μl buffer C —> centrifuge at 2.000 rpm for 2 min
    Note: Centrifugation times can vary, depending on sample volume, C18 phase tightness and sample composition.

  5. Elution:

    1. Place StageTips on top of the fresh Eppi
    2. Add 100μl buffer B to the StageTips
    3. Press the buffer down using a syringe until the StageTip material is wet (you see some liquid beneath the material)
    4. Incubate for 5 min at RT
    5. Push the buffer through the StageTip with a syringe or centrifugation
    6. Dry down the sample in the SpeedVac, ideally to a volume of approx. 2 μl
    7. Resuspend the sample in **30 μl of buffer A

  6. Perform peptide fluorimetric assay.

  7. Fill up with buffer A to reach a peptide concentration of 100ng/μl.

For storage, freeze in drawer -20°C or keep for short time in 4°C