Sep 01, 2025

Public workspaceEnrichment of phosphopeptides from lysate using TiO2

DOI
https://dx.doi.org/10.17504/protocols.io.rm7vzqdp2vx1/v1
  • Jasmin Jansen1,2,3
  • 1University of Konstanz, Department of Biology;
  • 2Aligning Science Across Parkinson's;
  • 3Konstanz Research School of Chemical Biology (KoRS-CB)
Jasmin Jansen
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DOI: https://dx.doi.org/10.17504/protocols.io.rm7vzqdp2vx1/v1
Protocol Citation: Jasmin Jansen 2025. Enrichment of phosphopeptides from lysate using TiO2. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzqdp2vx1/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 has worked in previous experiments.
Created: May 22, 2025
Last Modified: September 01, 2025
Protocol Integer ID: 218798
Keywords: ASAPCRN, phosphoenrichment, TiO2-MOAC, phosphopeptide, sample preparation for phosphopeptide enrichment, phosphopeptide enrichment, enrichment of phosphopeptide, enriched phosphopeptide, rich repeat kinase, phosphopeptide, phosphorylation, phosphorylation activity, cell lysate, wide interactomes of leucine, lysis of these cell, mass spectrometry, based mass spectrometry, proteome, lysi
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP))
Grant ID: ASAP-000519
Abstract
This protocol describes the sample preparation for phosphopeptide enrichment from lysed HEK293T cells that were transfected with TurboID, TurboID-LRRK1 or TurboID-LRRK2 and treated with MLi-2, IN04 or DMSO. It is meant to accompany the methods section and manuscript of "Probing the proteome-wide interactomes of Leucine-rich Repeat Kinases 1 and 2 and alterations in their phosphorylation activity".

For details on culturing and treatment of cells as well as lysis of these cells, please refer to Protocol "TurboID-phospho protocol" (doi: 10.17504/protocols.io.ewov1drpyvr2/v1) where these steps are explained in detail. This protocol is only meant to describe the sample processing from a cell lysate to enriched phosphopeptides ready to be measured by DIA-based mass spectrometry.
This protocol starts with the cell lysate obtained in the above mentioned protocol at step 14 and ends with enriched phosphopeptide that can be analysed by mass spectrometry according to steps 45ff of the same protocol.

All steps are described per sample.
Materials
ReagentcOmplete™, EDTA-free protease inhibitor cocktailRocheCatalog #11873580001
ReagentTris(2-carboxyethyl)phosphin -hydrochloridMerck MilliporeSigma (Sigma-Aldrich)Catalog #C4706
ReagentIodoacetamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #I1149-5G
ReagentTrypsin/Lys-C Mix, Mass Spec Grade, 5 x 20ugPromegaCatalog #V5073
ReagentSequencing Grade Modified TrypsinPromegaCatalog #V5113
ReagentHiPPR™ Detergent Removal Spin Column KitThermo FisherCatalog #88305
ReagentSep-Pak tC18 1 cc Vac Cartridge 50 mg Sorbent per CartridgeWatersCatalog #WAT054960
ReagentTitansphere TiO, 5 μm, 500 mgGL ScienceCatalog #020-75000
ReagentGlycolic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #124737
Reagent3M™ Empore™ C18 47 mm Extraction Disc Model 2215 20 pack 3 packs per case3M corporationCatalog #2215
ReagentLactic acid solution, 85 %Merck MilliporeSigma (Sigma-Aldrich)Catalog #252476

Protocol materials
ReagentTris(2-carboxyethyl)phosphin -hydrochloridMerck MilliporeSigma (Sigma-Aldrich)Catalog #C4706
ReagentIodoacetamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #I1149-5G
ReagentSequencing Grade Modified TrypsinPromegaCatalog #V5113
ReagentSep-Pak tC18 1 cc Vac Cartridge 50 mg Sorbent per CartridgeWatersCatalog #WAT054960
ReagentLactic acid solution, 85 %Merck MilliporeSigma (Sigma-Aldrich)Catalog #252476
ReagentGlycolic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #124737
ReagentcOmplete™, EDTA-free protease inhibitor cocktailRocheCatalog #11873580001
ReagentTrypsin/Lys-C Mix, Mass Spec Grade, 5 x 20ugPromegaCatalog #V5073
Reagent3M™ Empore™ C18 47 mm Extraction Disc Model 2215 20 pack 3 packs per case3M corporationCatalog #2215
ReagentHiPPR™ Detergent Removal Spin Column KitThermo FisherCatalog #88305
ReagentTitansphere TiO, 5 μm, 500 mgGL ScienceCatalog #020-75000
Troubleshooting
Safety warnings
After adding acetone to your proteins, always work on ice until you have dried the pellet. If your sample warms up during the precipitation process, you might lose proteins.

After performing the phosphopeptide enrichment, make sure that the exposition to basic pH is as short as possible because this can cause a loss of phosphorylations
Perform the enrichment shortly before measurement to keep native phosphorylation intact.
Before start
You will need 6 volumes of ice-cold acetone for 1 volume of your sample to perform the precipitation. Please calculate the total sample volume before you start and choose the right tube size for the precipitation.
Acetone-Protein precipitation
Thaw Amount200 µL cell lysate in NP-40 buffer (Concentration25 millimolar (mM) Tris-HCl Ph7.4 , Concentration150 millimolar (mM) NaCl, Concentration1 % (v/v) NP-40, Concentration5 millimolar (mM) MgCl2, Concentration1 millimolar (mM) Na3VO4, Concentration5 millimolar (mM) NaF, Concentration5 millimolar (mM) β-glycero phosphate, 1x ReagentcOmplete™, EDTA-free protease inhibitor cocktailRocheCatalog #11873580001 , Concentration1 millimolar (mM) DTT) or other lysis buffers at Temperature0 °C .

Add 1 volumes (here Amount200 µL ) of ice-cold acetone and mix sample.

Add 2 volumes (here Amount400 µL ) of ice-cold acetone and mix sample.

Add 3 volumes (here Amount600 µL ) of ice-cold acetone and mix sample.
Incubate at Temperature-80 °C for Duration00:15:00 , then at Temperature-20 °C for Duration01:30:00 .
Centrifuge precipitated samples for Duration00:10:00 at Centrifigation16000 x g, 4°C .

Afterwards, you should be able to see a whitish pellet of precipitated proteins (depending on the protein amount).

Carefully remove supernatant without disturbing the pellet.
Optionally, wash your pellet with ice-cold acetone one or two more times.
This depends on the downstream processing. Washing of the pellet can for example reduce the amount of detergents in the sample.
Dry the pellet at TemperatureRoom temperature with an open lid.

Protein reduction, alkylation and digestion
Resuspend protein pellet in Amount150 µL Concentration8 Molarity (M) urea.

Incubate for Duration00:15:00 at Temperature37 °C and Shaker800 rpm to resuspend proteins.

Add TCEP ReagentTris(2-carboxyethyl)phosphin -hydrochloridMerck MilliporeSigma (Sigma-Aldrich)Catalog #C4706 for Concentration3 millimolar (mM) final concentration to reduce cysteines.
Incubate for Duration00:30:00 at Temperature37 °C and Shaker650 rpm .
Add IAA (ReagentIodoacetamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #I1149-5G ) to Concentration6 millimolar (mM) final concentration to alkylate cysteines.
Incubate for Duration00:30:00 at TemperatureRoom temperature and Shaker650 rpm in the dark.
Dilute urea to Concentration4 Molarity (M) using Concentration50 millimolar (mM) ammonium bicarbonate.
Add Amount1 µg ReagentTrypsin/Lys-C Mix, Mass Spec Grade, 5 x 20ugPromegaCatalog #V5073 to sample.
Digestion
Incubate for Duration03:30:00 at Temperature37 °C and Shaker650 rpm .
Dilute urea concentration to Concentration1 Molarity (M) using Concentration50 millimolar (mM) ammonium bicarbonate.
Add Amount2 µg ReagentSequencing Grade Modified TrypsinPromegaCatalog #V5113 .
Digestion
Incubate for Duration18:00:00 at Temperature37 °C and Shaker650 rpm .
Overnight
Remove residual detergents with the ReagentHiPPR™ Detergent Removal Spin Column KitThermo FisherCatalog #88305 (Amount200 µL per sample ) according to the manufacturer's instructions.
Acidify flow-through by adding formic acid to a final concentration of Concentration2 % (v/v) .
After mixing, check if Ph2 or lower. This is important since acidic pH stops trypsin digestion and is crucial for a successful C18 desalting.
Critical
Add Concentration1 % (v/v) acetonitrile (ACN) and desalt your sample using ReagentSep-Pak tC18 1 cc Vac Cartridge 50 mg Sorbent per CartridgeWatersCatalog #WAT054960 and a vacuum manifold.
Wet cartridges with Amount1 mL pure ACN.

Equilibrate cartridges with Amount2 mL Concentration1 % (v/v) ACN, Concentration0.1 % (v/v) formic acid.
Add sample and apply it slowly to the C18 resin. Do not open valve fully to load sample slowly.
Wash peptides with Amount2 mL Concentration1 % (v/v) ACN, Concentration0.1 % (v/v) formic acid.
Elute peptides into low-binding 1.5 mL tube with Amount1 mL Concentration50 % (v/v) ACN, Concentration0.1 % (v/v) formic acid.
Dry desalted peptides in a vaccuum concentrator.
Pause
Phosphopeptide enrichment
Weigh Amount2 mg beads per sample (if ~ Amount200 µg peptide input) ReagentTitansphere TiO, 5 μm, 500 mgGL ScienceCatalog #020-75000 and resuspend in Amount10 µL per 1 mg beads Loading buffer 1 (Concentration0.1 Molarity (M) ReagentGlycolic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #124737 , Concentration70 % (v/v) ACN, Concentration5 % (v/v) trifluoroacetic acid (TFA)).

To minimize unspecific binding, incubate TiO2 beads in Loading buffer 1 for Duration00:20:00 at TemperatureRoom temperature and Shaker1200 rpm prior to peptide incubation.
Resuspend approx. Amount200 µg of dried peptides in Amount200 µL Loading buffer 1.
Incubate for Duration00:10:00 at Temperature37 °C and Shaker650 rpm for proper resuspension.

Add equilibrated beads to peptides at a peptide:bead ratio of 1:10.
Incubate for Duration00:20:00 at TemperatureRoom temperature on rolling incubator.
Prepare self-made, single-layered C8 Stage tips. See
Citation
Rappsilber J, Mann M, Ishihama Y (2006). Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips.
https://doi.org/
LINK
for details.
Use Reagent3M™ Empore™ C18 47 mm Extraction Disc Model 2215 20 pack 3 packs per case3M corporationCatalog #2215 to make the tips.
Equilibrate C8 Stage tips with Loading buffer 1.
Settle incubated TiO2 beads by centrifugation Centrifigation10000 x g for Duration00:02:00 .
Transfer Amount150 µL of supernatant to fresh low-binding tube and keep for second enrichment step, see Go to .
Use residual supernatant to transfer beads to equilibrated C8 Stage tips.
Centrifuge for Duration00:02:00 at Centrifigation500 x g and add flow-through to supernatant fraction from step Go to .
Wash beads that were retained by C8 Stage tips with Amount50 µL Wash buffer 1 (Concentration80 % (v/v) ACN, Concentration1 % (v/v) TFA) and centrifuge at Centrifigation500 x g for Duration00:02:00 .
Wash beads with Amount50 µL Wash buffer 2 (Concentration10 % (v/v) ACN, Concentration0.2 % (v/v) TFA) and centrifuge at Centrifigation500 x g for Duration00:02:00 . Transfer tips to new vial.
Prepare low-binding tubes with Amount60 µL Concentration10 % (v/v) formic acid for phosphopeptide elutions to directly acidify them and minimize loss of phosphorlyation by ammonium hydroxide.
Elute bound phosphopeptides with Amount30 µL Elution buffer 1 (Concentration1 % (v/v) NH4OH) by centrifugation at Centrifigation500 x g for Duration00:02:00 and directly transfer elution to prepared tubes (Go to ).
Elute residual bound phosphopeptides with Amount30 µL Elution buffer 2 (Concentration5 % (v/v) NH4OH, Concentration25 % (v/v) ACN) by centrifugation at Centrifigation500 x g for Duration00:02:00 and directly transfer elution to prepared tubes (Go to ).
Repeat phosphopeptide protocol with retained supernatant from Go to and repeat protocol with Loading buffer 2 (Concentration20 % (v/v) ReagentLactic acid solution, 85 %Merck MilliporeSigma (Sigma-Aldrich)Catalog #252476 , Concentration70 % (v/v) ACN, Concentration5 % (v/v) TFA) instead of Loading buffer 1.

This second enrichment step maximizes yield of phosphopeptide by repeating the enrichment and changing the specificity by using lactic acid instead of glycolic acid.
Combine phosphopeptide elutions and dry them by vaccuum evaporation.
Samples are now ready for mass spectrometric analysis. Please refer to protocol "TurboID-phospho protocol" (dx.doi.org/10.17504/protocols.io.ewov1drpyvr2/v1) for details.
Acknowledgements
This research was funded by Aligning Science Across Parkinson’s (grant ASAP-000519) through the Michael J. Fox Foundation for Parkinson’s Research (MJFF). This work was also supported by funding of the German Research Foundation (DFG, 496470458 and 516836828).