Mar 29, 2024
Proteolytic Peptides from Conditioned Media Desalting with C18 Hydrophilic–Lipophilic Balance (HLB) Cartridges
- Joanna Bons1,
- J P Rose1,
- M A Watson1,
- B Schilling1
- 1Buck Institute for Research on Aging
Protocol Citation: Joanna Bons, J P Rose, M A Watson, B Schilling 2024. Proteolytic Peptides from Conditioned Media Desalting with C18 Hydrophilic–Lipophilic Balance (HLB) Cartridges . protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lywdzpvx9/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: March 25, 2024
Last Modified: March 29, 2024
Protocol Integer ID: 97347
Keywords: Desalting, HLB, Peptides, Proteomics, Mass Spectrometry, Conditioned Media, Secretome, SASP
Abstract
Desalting of proteolytic peptides from conditioned media elution using C18 Hydrophilic–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.
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 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.