Proteolytic Peptides from Conditioned Media Desalting with C18 Hydrophilic–Lipophilic Balance (HLB) Cartridges
J P Rose, M A Watson, B Schilling, Joanna Bons
Abstract
Desalting of proteolytic peptides from conditioned media elution using C18Hydrophilic–Lipophilic Balance (HLB) Cartridges in preparation for downstream proteomic profiling.
For desalting of proteolytic peptides from conditioned media Step 4 is modified as we typically reconstitute in an appropriate volume of 0.2% FA rather than by weight/volume due to the input material for digestion also being based on a proportion of the volume of concentrated conditioned media.
Steps
Centrifuge the samples at 1,850 x g for 5 minutes at room temperature to pellet insoluble material.
Desalt the samples using Oasis HLB solid-phase extraction cartridges placed on top of a vacuum manifold as follows:
Condition each cartridge two times with 800 µL of the condition buffer (50% ACN, 0.2% FA).
Equilibrate each cartridge three times with 800 µL of the equilibration buffer (0.2% FA).
Load the peptide samples.
Wash each cartridge three times with 800 µL of the wash buffer (0.2% FA).
Elute peptides with 800 µL of the elution buffer (50% ACN, 0.2% FA) followed by a further 400 µL of the same elution buffer.
Vacuum dry the eluted peptide solution in a centrifugal vacuum concentrator.
Reconstitute the dried peptides in an appropriate volume of 0.2% FA and thoroughly mix the solution.
Centrifuge at 12,000 x g at room temperature for 2 min, and store at -20°C.
