Oct 20, 2023
Proteolytic Peptide Desalting with C18 Hydrophilic–Lipophilic Balance (HLB) Cartridges
- J P Rose1,
- M A Watson1,
- B Schilling1,
- Joanna Bons1
- 1Buck Institute for Research on Aging
Protocol Citation: J P Rose, M A Watson, B Schilling, Joanna Bons 2023. Proteolytic Peptide Desalting with C18 Hydrophilic–Lipophilic Balance (HLB) Cartridges. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4q382vo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 20, 2023
Last Modified: October 20, 2023
Protocol Integer ID: 89689
Keywords: Desalting, HLB, Peptides, Proteomics, Mass Spectrometry
Abstract
Desalting of proteolytic peptide elution using C18 Hydrophilic–Lipophilic Balance (HLB) Cartridges in preparation for downstream proteomic profiling.
Materials
- Oasis HLB 1cc cartridges, 10 mg sorbent (Waters, Cat. #WAT058882)
- Vacuum manifold
- Centrifuge
- Centrifugal vacuum concentrator
- 1.5-mL microcentrifuge tubes
- Condition buffer/Elution buffer: 50% acetonitrile (ACN), 0.2% formic acid (FA) in water
- Equilibration buffer/Wash buffer: 0.2% formic acid (FA) in water
- HPLC-grade water
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 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.