laboratory
Desalting strategy for protein content > 20 μg

Chromabond C18-columns desalting protocol

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.

Solvents

  • C18 Macherey-Nagel Chromabond C18 columns

  • 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. Place the columns on the appropriate stand or in the 2 ml Eppendorf tubes1

  2. Equilibrate the C18 columns:

    1. Add 600 µl acetonitrile to the C18 columns and centrifuge at 1.000 rpm for 1 min2

    2. Add 600 µl buffer B to the C18 columns and centrifuge at 1.000 rpm for 1 min

    3. Add 600 µl buffer A to the C18 columns and centrifuge at 1.000 rpm for 1 min

    4. Add 600 µl buffer A to the C18 columns and centrifuge at 1.000 rpm for 1 min

  3. Load the samples onto the C18 columns

  4. Wash the C18 columns with 600 µl buffer C

  5. Elution:

    1. Place C18 columns on top of the fresh 2 ml Eppendorf tube

    2. Add 200 µl buffer B to the C18 columns

    3. Press the buffer down using a syringe until the C18 column material is wet

    4. Incubate for 5 min at RT

    5. Push the buffer through the C18 columns with a syringe or centrifugation

    6. Add 100 µl buffer B to the C18 columns and repeat the elution

    7. Dry down the sample in the SpeedVac, ideally to a volume of approx. 2 µl

    8. 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

Footnotes

  1. Dependent on the available equipment, all subsequent steps you can perform either in a big centrifuge with plate holders and special adapters, normal benchtop centrifuge or just use a gravitational force and wait for the liquid to drop through the column.↩︎

  2. The speed is arbitrary. We aim to slowly push the reagents through the column and try not to overdry it.↩︎