Proteolytic Peptide Desalting with C18 Hydrophilic–Lipophilic Balance (HLB) Cartridges

Abstract

Desalting of proteolytic peptide elution using C₁₈ Hydrophilic–Lipophilic Balance (HLB) Cartridges in preparation for downstream proteomic profiling.

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:

2.1.

Condition each cartridge two times with 800 µL of the condition buffer (50% ACN, 0.2% FA).

2.2.

Equilibrate each cartridge three times with 800 µL of the equilibration buffer (0.2% FA).

2.3.

Load the peptide samples.

2.4.

Wash each cartridge three times with 800 µL of the wash buffer (0.2% FA).

2.5.

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 0.2% FA for a final protein concentration of 1 µg/µL and thoroughly mix the solution.

Centrifuge at 12,000 x g at room temperature for min, and store at -20°C.