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SPE Protein/Peptide Clean-Up Protocol

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PREPARATION NOTES

The maximum amount of protein/peptides to load on the SPE Cartridge is 50 µg.

Reagents required in this protocol for bottom-up workflows:
100% methanol, 50% acetonitrile/0.1 % TFA, 5% acetonitrile/0.1 % TFA, 100 % acetonitrile

Reagents required in this protocol for top-down workflows:
100% methanol, 50% acetonitrile/0.1 % TFA, 5% acetonitrile/0.1 % TFA, 100 % acetonitrile, 30% isopropanol/42% formic acid, 40% isopropanol/36% formic acid

PROTOCOL

The SPE Cartridge must first be primed with the attached Priming Cartridge.

Avoid excessive spinning, a few microliters of the final priming solution left behind is permissible during priming, loading, and wash. The final elution step can be spun to complete dryness.

PRIME (It is advisable to prime the SPE Cartridge just prior to use.)

  • Add 300 µL 100% methanol; centrifuge 400 ×g (2000 rpm) ×2 minutes. Discard flow through.
  • Followed by 300 µL 50% acetonitrile/0.1% TFA, centrifuge 400 ×g (2000 rpm) ×2 minutes. Discard flow-through.
  • Finally, 300 µL 5% acetonitrile/0.1% TFA centrifuge 400 ×g (2000 rpm) ×2 minutes. Discard flow-through.
  • Remove SPE Cartridge from the Priming Cartridge immediately and attach it to the base of the Filtration Cartridge containing the protein.

LOAD

  • Add 30 µL clean acetonitrile to your sample (bringing the final concentration of acetonitrile to 5%).
  • Spin the Filtration Cartridge with the SPE Cartridge attached at 800 ×g (3000 rpm) ×5 minutes. Ensure that no more than a few microliters of solvent remains in the Filtration Cartridge, if so spin again.
  • OPTIONAL: Reload the eluent into the SPE Cartridge 800 ×g (3000 rpm) ×5 minutes. This second pass can improve SPE retention.

WASH

Add 300 µL 5% acetonitrile, 0.1% TFA in water. Spin at 2000 ×g (4500 rpm) ×2 minutes. Discard flow through.

ELUTE

Digested Peptides:

  • Add 300 μL of 50% acetonitrile, 0.1% TFA, water. Spin 2500 ×g (5000 rpm) ×5 minutes.
  • Retain the eluent.

OR

Intact protein:

  • Add 300 μL of 30% isopropanol/42% formic acid.
  • Followed by 300 μL of 40% isopropanol/36 % formic acid.
  • Centrifuge 2500 ×g (5000 rpm) ×5 minutes.
  • Pool eluates.

Vacuum dry and reconstitute in desired LC-MS/MS solvent.