Oct 22, 2025
Denaturing On-bead Digestion for LC-MS/MS
- Katja Piltti1,
- Clinton Yu1
- 1University of California, Irvine
Protocol Citation: Katja Piltti, Clinton Yu 2025. Denaturing On-bead Digestion for LC-MS/MS. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6zp91gqe/v1
Manuscript citation:
Piltti KM et al. C1q drives neural stem cell quiescence by regulating cell cycle and metabolism through BAI1. Nature Communications (In press)
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 08, 2025
Last Modified: October 22, 2025
Protocol Integer ID: 229372
Keywords: On-bead digestion, Denaturing protein digestion, Pull-down assay, Protein purification, Magnetic bead pull-down, Affinity purification, NHS-SS-Biotin labeling, DSP crosslinker, Streptavidin magnetic beads, Biotinylation-based protein capture, Crosslinked protein complexes, Proteomics sample prep, LC-MS/MS preparation, bead digestion for protein purification, bead digestion for lc, protein purification, protein complexes for unbiased forward screen, bead digestion, using magnetic bead, interacting intracellular protein, compatible with the cell membrane, magnetic bead, using nanolc, protein, biotin, protein complex, cell membrane
Funders Acknowledgements:
Wings for Life
Grant ID: WFL-US-01/18
Abstract
A denaturing on-bead digestion for protein purification after pull-down using magnetic beads. The protocol is compatible with the cell membrane-permeable Ez-link NHS-SS-Biotin (ThermoScientific, PI21441) labeling and cell membrane-permeable DSP (ThermoScientific, PI22586). Step-by-step instructions for the pull-down are available in "Co-Immunoprecipitation/Pull-Down of C1q-Interacting Intracellular Proteins/Protein Complexes for Unbiased Forward Screen Using NanoLC-MS/MS"-protocol.
Guidelines
1. Wash beads with HPLC grade H2O and remove the supernatant.
2. Add 1 bed volume of 2M urea with 25 mM ammonium bicarbonate in water to the magnetic beads with protein.
Note: Bed volume refers to the volume of the resin or magnetic beads. For example, if you have 100 µL of a 50% slurry, the bed volume is 50 µL once settled.
3. Add Dithiothreitol (DTT) to a final concentration of 2 mM and incubate for 15 minutes at 37°C.
4. Add iodoacetamide to a final concentration of 4 mM and incubate for 30 minutes in the dark at room temperature.
5. Add DTT to a final concentration of 2 mM and incubate for 15 minutes at 37°C to quench.
6. Add 1 bed volume of 25 mM ammonium bicarbonate to dilute the urea to a final concentration of 1M.
7. Add trypsin to a final concentration of 20 ng/µL. Incubate at 37°C for 8 hours to overnight. *Dissolved in 50 mM acetic acid.
8. Add formic acid to 0.1% to stop the digestion.
9. Collect the supernatant from the beads for a c18 cleanup.
Materials
- HPLC grade H2O (Fisher Scientific, Cat# W5-4)
- 2M urea (Fisher Scientific, Cat# BP169-500)
- Ammonium bicarbonate (Sigma-Aldrich, Cat# 09830)
- Dithiothreitol (DTT) (Fisher Scientific, Cat# BP172-25)
- Iodoacetamide (Sigma-Aldrich, Cat# I6125)
- Trypsin (Fisher Scientific, Cat# PRV5111)
- 50 mM acetic acid (Millipore-Sigma, Cat# 5330010050)
- Formic acid (Fisher Scientific, Cat# 60-006-17)
Troubleshooting
Proceed with the following steps for protein denaturation, reduction, and alkylation:
Wash beads with HPLC-grade H2O (Fisher Scientific, Cat# W5-4) and remove the supernatant.
Add 1 bed volume of 2M urea (Fisher Scientific, Cat# BP169-500) with 25 mM ammonium bicarbonate (Sigma-Aldrich, Cat# 09830) in water to the magnetic beads with protein.
Note: Bed volume refers to the volume of the resin or magnetic beads. For example, if you have 100 µL of a 50% slurry, the bed volume is 50 µL once settled.
Add Dithiothreitol (DTT) (Fisher Scientific, Cat# BP172-25) to a final concentration of 2 mM and incubate for 15 minutes at 37°C.
Add iodoacetamide (Sigma-Aldrich, Cat# I6125) to a final concentration of 4 mM and incubate for 30 minutes in the dark at room temperature.
Add DTT to a final concentration of 2 mM and incubate for 15 minutes at 37°C to quench.
Add 1 bed volume of 25 mM ammonium bicarbonate to dilute the urea to a final concentration of 1M.
Add trypsin* (Fisher Scientific, Cat# PRV5111) to a final concentration of 20 ng/µL. Incubate at 37°C for 8 hours to overnight. *Dissolved in 50 mM acetic acid (Millipore-Sigma, Cat# 5330010050).
Add formic acid (Fisher Scientific, Cat# 60-006-17) to 0.1% to stop the digestion.
Collect the supernatant from the beads for a C18 cleanup. See Sample Desalting Using OMIX C18 Tips for LC-MC-protocol for details.