Sep 01, 2025
TurboID-phosphoenrichment workflow protocol
- Jasmin Jansen1,2,3
- 1University of Konstanz, Department of Biology;
- 2Aligning Science Across Parkinson's Disease;
- 3Konstanz Research School of Chemical Biology (KoRS-CB)
Protocol Citation: Jasmin Jansen 2025. TurboID-phosphoenrichment workflow protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1drpyvr2/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 our previous experiments.
Created: March 04, 2025
Last Modified: September 01, 2025
Protocol Integer ID: 123769
Keywords: ASAPCRN, TurboID, Phosphoenrichment, quantitative proteomics, DIA-MS, phosphoproteomics, phosphopeptide enrichment to mass spectrometric analysis, rich repeat kinase, phosphosites of this interactome, wide interactomes of leucine, phosphopeptide enrichment, phosphorylation activity, phosphorylation, cell culture over turboid, mass spectrometric analysis, changed phosphosite, streptavidin enrichment, based phosphoenrichment, proteome, comprehensive dataset of interactor, wide interactome, interactome
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000519
German Research Foundation
Grant ID: 496470458
German Research Foundation
Grant ID: 516836828
Abstract
This protocol is meant to accompany the manuscript "Probing the proteome-wide interactomes of Leucine-rich Repeat Kinases 1 and 2 and alterations in their phosphorylation activity" and its methods section.
The protocol describes all steps of the experimental workflow starting from cell culture over TurboID, streptavidin enrichment and phosphopeptide enrichment to mass spectrometric analysis using Data-Independent acquisition (DIA)-based quantification and data analysis.
The aim is to get a comprehensive dataset of interactors together with changed phosphosites of this interactome based on TurboID (Cho et al., 2020) and TiO2-based phosphoenrichment.
All steps are described per sample.
Guidelines
When preparing samples for mass spectrometry, work carefully, always wear appropriate gloves to minimize sample contamination that impedes your analysis. For downstream applications like mass spectrometry, it is crucial to wash cell pellets carefully to minimize protein contamination through fetal bovine serum.
For cell culture, please check your cell line(s) routinely for mycoplasma and other contaminations to avoid artefacts introduced thereby.
Materials
Anti-LRRK2 (phospho S935) antibody [UDD2 10(12)]AbcamCatalog #ab133450 Primary Antibody anti-LRRK2 c41-2 AbcamCatalog #ab133474 Lactic acid solution, 85 %Merck MilliporeSigma (Sigma-Aldrich)Catalog #252476 Glycolic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #124737 Titansphere TiO, 5 μm, 500 mgGL ScienceCatalog #020-75000 Sequencing Grade Modified TrypsinPromegaCatalog #V5113
Equipment
Sep-Pak tC18 1 cc Vac Cartridge, 50 mg Sorbent per Cartridge, 37 - 55 µm
NAME
C18 column
TYPE
Sep-Pak
BRAND
WAT054960
SKU
LINK
Equipment
Pierce™ C18-Spin columns
NAME
C18 column
TYPE
Pierce
BRAND
84850
SKU
LINK
Trypsin/Lys-C Mix, Mass Spec Grade, 5 x 20ugPromegaCatalog #V5073 IodoacetamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #I1149-5G Tris(2-carboxyethyl)phosphin -hydrochloridMerck MilliporeSigma (Sigma-Aldrich)Catalog #C4706 Pierce™ Streptavidin Magnetic BeadsThermo FisherCatalog #88817 cOmplete™, EDTA-free protease inhibitor cocktailRocheCatalog #11873580001 Formaldehyede 37% solutionCarl RothCatalog #7398 Dulbeccos Phosphate-buffered salineGibco - Thermo Fisher ScientificCatalog #21300058 IN04AOBIOUS IncCatalog #AOB13355 MLi-2Merck MilliporeSigma (Sigma-Aldrich)Catalog #SML3101 BiotinMerck MilliporeSigma (Sigma-Aldrich)Catalog #B4639 Polyethylenimine, Linear, MW 25000, Transfection GradePolysciences, Inc.Catalog #23966 Opti-MEM™ I Reduced Serum MediumThermo FisherCatalog #31985070 Dulbeccos modified eagle medium (DMEM), high glucoseGibco - Thermo Fisher ScientificCatalog #41965062
Fetal bovine serum (FBS) SuperiorMerck MilliporeSigma (Sigma-Aldrich)Catalog #S0615
DMSOSupelcoCatalog #1.09678.0100 HRP Anti-Rabbit IgGDianovaCatalog #AKC080 Anti-RAB10 (phospho T73) antibody [MJF-R21]AbcamCatalog #ab230261 Recombinant Anti-RAB10 antibody [MJF-R23]AbcamCatalog #ab237703
HiPPR™ Detergent Removal Spin Column KitThermo FisherCatalog #88305 Sep-Pak tC18 1 cc Vac Cartridge 50 mg Sorbent per CartridgeWatersCatalog #WAT054960 3M™ Empore™ C18 47 mm Extraction Disc Model 2215 20 pack 3 packs per case3M corporationCatalog #2215
Acclaim™ PepMap™ 100 C18 HPLC ColumnsThermo ScientificCatalog #164945
Equipment
Concentrator Plus
NAME
Centrifugal vacuum concentrator
TYPE
Eppendorf
BRAND
5305000568
SKU
LINK
Protocol materials
Dulbeccos modified eagle medium (DMEM), high glucoseGibco - Thermo Fisher ScientificCatalog #41965062
Fetal bovine serum (FBS) SuperiorMerck MilliporeSigma (Sigma-Aldrich)Catalog #S0615
Dulbeccos Phosphate-buffered salineGibco - Thermo Fisher ScientificCatalog #21300058
Formaldehyede 37% solutionCarl RothCatalog #7398
BiotinMerck MilliporeSigma (Sigma-Aldrich)Catalog #B4639
cOmplete™, EDTA-free protease inhibitor cocktailRocheCatalog #11873580001
Tris(2-carboxyethyl)phosphin -hydrochloridMerck MilliporeSigma (Sigma-Aldrich)Catalog #C4706
IodoacetamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #I1149-5G
Trypsin/Lys-C Mix, Mass Spec Grade, 5 x 20ugPromegaCatalog #V5073
HiPPR™ Detergent Removal Spin Column KitThermo FisherCatalog #88305
Acclaim™ PepMap™ 100 C18 HPLC ColumnsThermo ScientificCatalog #164945
Recombinant Anti-RAB10 antibody [MJF-R23]AbcamCatalog #ab237703
3M™ Empore™ C18 47 mm Extraction Disc Model 2215 20 pack 3 packs per case3M corporationCatalog #2215
Glycolic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #124737
Titansphere TiO, 5 μm, 500 mgGL ScienceCatalog #020-75000
Sequencing Grade Modified TrypsinPromegaCatalog #V5113
Primary Antibody anti-LRRK2 c41-2 AbcamCatalog #ab133474
IN04AOBIOUS IncCatalog #AOB13355
Lactic acid solution, 85 %Merck MilliporeSigma (Sigma-Aldrich)Catalog #252476
Anti-LRRK2 (phospho S935) antibody [UDD2 10(12)]AbcamCatalog #ab133450
MLi-2Merck MilliporeSigma (Sigma-Aldrich)Catalog #SML3101
Polyethylenimine, Linear, MW 25000, Transfection GradePolysciences, Inc.Catalog #23966
HRP Anti-Rabbit IgGDianovaCatalog #AKC080
Sep-Pak tC18 1 cc Vac Cartridge 50 mg Sorbent per CartridgeWatersCatalog #WAT054960
Pierce™ Streptavidin Magnetic BeadsThermo FisherCatalog #88817
DMSOSupelcoCatalog #1.09678.0100
Anti-RAB10 (phospho T73) antibody [MJF-R21]AbcamCatalog #ab230261
Opti-MEM™ I Reduced Serum MediumThermo FisherCatalog #31985070
Troubleshooting
Safety warnings
Before performing the experiment, please optimize the incubation of biotin with TurboID-transfected cells to avoid overlabelling. This can be done by SDS-PAGE and Western blotting or mass spectrometry.
Please do not skip the detergent removal step as it is crucial for an optimal mass spectrometric analysis. Detergents like NP-40 will interfer with C18 nano-HPLC and lead to non-optimal resolution of peptides. Also, they can contaminate the mass spectrometers and cause ion suppression.
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
Please read the protocol carefully before you start the experiment to prepare all materials and methods and plan time and resources accordingly.
Cell culture
1d 2h 15m
HEK293T wt cells are cultured in 10 cm dishes in Dulbeccos modified eagle medium (DMEM), high glucoseGibco - Thermo Fisher ScientificCatalog #41965062 with 10 % (v/v) Fetal bovine serum (FBS) SuperiorMerck MilliporeSigma (Sigma-Aldrich)Catalog #S0615 (hereafter "medium") at 37 °C , 95 % and 5 % (v/v) CO2 and passaged upon confluency.
For transient transfection and proximity labeling, cells were grown in 15 cm Petri dishes to 50-60 % confluency. For 1 replicate, 4x 15 cm dishes were used to have enough material for the interactome enrichment.
Transient transfection
Change medium and add 12.5 mL fresh medium to each plate.
For one replicate (4x 15cm dish), mix7.5 µg of plasmid carrying TurboID-fusion construct with 5 mL Opti-MEM™ I Reduced Serum MediumThermo FisherCatalog #31985070 . Add 90 µL Polyethylenimine, Linear, MW 25000, Transfection GradePolysciences, Inc.Catalog #23966 (1 mg/mL stock solution).
Mix well by pipetting.
Incubate for 00:15:00 at Room temperature .
15m
Add 1.25 mL of the mix to each of the four plates. Tilt the plates and carefully add dropwise into medium on the side to not disturb adherent cells.
Incubate for 24:00:00 at 37 °C , 95 % and 5 % (v/v) CO2 .
1d
Addition of inhibitors
Change medium and add DMSO/inhibitors with this medium change
Add 5 µL of either 10 millimolar (mM) MLi-2Merck MilliporeSigma (Sigma-Aldrich)Catalog #SML3101 , 50 millimolar (mM) IN04AOBIOUS IncCatalog #AOB13355 (both in DMSO) or DMSO to 50 mL medium for 1 micromolar (µM) final MLi-2 or 5 micromolar (µM) final IN04 inhibitor concentration.
Add 12.5 mL of the prepared 50 mL mix to each of the four plates of 1 replicate.
Incubate for 01:00:00 at 37 °C , 95 % and 5 % (v/v) CO2
1h
TurboID proximity labelling
1h
Add 6.25 µL 100 millimolar (mM) BiotinMerck MilliporeSigma (Sigma-Aldrich)Catalog #B4639 in DMSO carefully to each of the plates to start proximity labelling.
Incubate for 01:00:00 at 37 °C , 95 % and 5 % (v/v) CO2 .
1h
Harvest
20m
Adherent cells were washed carefully with 2x Dulbeccos Phosphate-buffered salineGibco - Thermo Fisher ScientificCatalog #21300058 that was equilibrated to 37 °C prior to washing.
Add 5 mL warm PBS with 0.025 % (v/v) Formaldehyede 37% solutionCarl RothCatalog #7398 to each plate.
Incubate for 00:10:00 at Room temperature .
10m
Remove liquid carefully
Add 5 mL warm 50 millimolar (mM) Tris-HCl 7.4 to the adherent cells.
Incubate for 00:05:00 at Room temperature
5m
Remove liquid carefully.
Place cell dishes on ice and harvest cells with 10 mL ice-cold 1xDulbeccos Phosphate-buffered salineGibco - Thermo Fisher ScientificCatalog #21300058 per plate.
Transfer ice-cold cell suspension to 50 mL Falcon tubes and pool the suspensions from 4x 15 cm dishes for each replicate (same transfection/inhibitor/biotin mix for each of the 15 cm dishes).
Wash cells twice with 10 mL ice-cold 1xDulbeccos Phosphate-buffered salineGibco - Thermo Fisher ScientificCatalog #21300058 .
Pellet cells by centrifugation 800 x g, 4°C for 00:05:00 in between.
Pellet cells by centrifugation 800 x g, 4°C for 00:05:00 and snap freeze in liquid N2.
5m
Store cell pellets at-80 °C until further processing.
Cell lysis
35m
Place frozen cell pellets on ice. When working with cells and cell lysate please always work on ice until proteins are denatured or the protocol indicates to do otherwise.
Resuspend cell pellets in 16 mL lysis buffer (25 millimolar (mM) Tris-HCl 7.4 , 150 millimolar (mM) NaCl, 1 % (v/v) NP-40, 5 millimolar (mM) MgCl2, 1 millimolar (mM) Na3VO4, 5 millimolar (mM) NaF, 5 millimolar (mM) β-glycero phosphate, 1x cOmplete™, EDTA-free protease inhibitor cocktailRocheCatalog #11873580001 , 1 millimolar (mM) DTT).
Incubate for 00:15:00 at 4 °C while gently mixing/rolling.
15m
Pellet cell debris by centrifugation at 16000 x g, 4°C for 00:20:00 .
20m
Take supernatant and continue with streptavidin enrichment. Discard the pellet.
Streptavidin enrichment
20h 3m
Equilibrate 0.5 mL Pierce™ Streptavidin Magnetic BeadsThermo FisherCatalog #88817 (5 mg per sample ) with 3x 500 µL lysis buffer in 1.5 mL tube using a magnetic rack.
3m
Add the equilibrated beads to the supernatant from (see previous section).
Incubate for 20:00:00 at 4 °C and gentle agitation to allow binding of biotinylated proteins to streptavidin beads.
20h
Pellet beads by centrifugation 2000 x g, 4°C for 00:15:00 .
Use a magnet to carefully decant "flow-through" of streptavidin beads without disturbing the bead pellet.
If necessary, repeat centrifugation to retain as many beads as possible.
Resuspend beads with 1 mL lysis buffer and transfer to a fresh 2 mL reaction tube.
Wash beads with 1 mL lysis buffer using a magnetic rack.
Repeat washing step.
On-bead digestion
22h
Equilibrate streptavidin beads twice with 1 mL 50 millimolar (mM) ammonium bicarbonate to prepare samples for on-bead digestion.
Resuspend beads in 150 µL 8 Molarity (M) urea.
Add TCEP Tris(2-carboxyethyl)phosphin -hydrochloridMerck MilliporeSigma (Sigma-Aldrich)Catalog #C4706 for 3 millimolar (mM) final concentration to reduce cysteines.
Incubate for 00:30:00 at 37 °C and 650 rpm .
Add IAA (IodoacetamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #I1149-5G ) to 6 millimolar (mM) final concentration to alkylate cysteines.
Incubate for 00:30:00 at Room temperature and 650 rpm in the dark.
30m
Dilute urea to 4 Molarity (M) using 50 millimolar (mM) ammonium bicarbonate.
Add 3 µg Trypsin/Lys-C Mix, Mass Spec Grade, 5 x 20ugPromegaCatalog #V5073 to sample.
Incubate for 03:30:00 at 37 °C and 650 rpm .
3h 30m
Dilute urea concentration to 1 Molarity (M) using 50 millimolar (mM) ammonium bicarbonate.
Add 1.5 µg Sequencing Grade Modified TrypsinPromegaCatalog #V5113 .
Incubate for 18:00:00 at 37 °C and 650 rpm .
18h
Remove residual detergents with the HiPPR™ Detergent Removal Spin Column KitThermo FisherCatalog #88305 (200 µg per sample ) according to the manufacturer's instructions.
Acidify flow-through by adding formic acid to a final concentration of 2 % (v/v) .
After mixing, check if 2 or lower. This is important since acidic pH stops trypsin digestion and is crucial for a successful C18 desalting.
Add 1 % (v/v) acetonitrile (ACN) final concentration and desalt your sample using Sep-Pak tC18 1 cc Vac Cartridge 50 mg Sorbent per CartridgeWatersCatalog #WAT054960 and a vacuum manifold.
Wet cartridges with 1 mL pure ACN.
Equilibrate cartridges with 2 mL 1 % (v/v) ACN, 0.1 % (v/v) formic acid.
Add sample and apply it slowly to the C18 resin. Don't open the valve fully to apply slowly.
Wash peptides with 2 mL 1 % (v/v) ACN, 0.1 % (v/v) formic acid.
Elute peptides into low-binding 1.5 mL tube with 1 mL 50 % (v/v) ACN, 0.1 % (v/v) formic acid.
For further analysis, split sample for peptide and phosphopeptide analysis. Therefore, use 10 % of sample for "normal" peptide analysis (Fraction 1), and 90 % of sample for subsequent phosphopeptide enrichment and phosphopeptide analysis (Fraction 2).
Dry both fractions in a
Equipment
Concentrator Plus
NAME
Centrifugal vacuum concentrator
TYPE
Eppendorf
BRAND
5305000568
SKU
LINK
.
For Fraction 1, continue directly to "Mass spectrometric analysis".
For Fraction 2, continue with "Phosphopeptide enrichment".
Phosphopeptide enrichment
1h 2m
Weigh 0.5 mg beads per sample (if 50 µg peptides) Titansphere TiO, 5 μm, 500 mgGL ScienceCatalog #020-75000 and resuspend in Loading buffer 1 (0.1 Molarity (M) Glycolic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #124737 , 70 % (v/v) ACN, 5 % (v/v) trifluoroacetic acid (TFA)) so that 10 µL per 1 mg beads .
To minimize unspecific binding, incubate TiO2 beads in Loading buffer 1 for 00:20:00 at Room temperature and 1200 rpm prior to peptide incubation
20m
Resuspend approx. 50 µg of dried peptides in 200 µL Loading buffer 1.
Incubate for 00:10:00 at 37 °C for proper resuspension.
10m
Add equilibrated beads to peptides at a peptide:bead ratio of 1:10. If prepared as described, add 5 µL per sample.
Incubate for 00:20:00 at Room temperature in rolling incubator.
20m
Prepare self-made, single-layered C8 Stage tips. See
Citation
LINK
for details.
Use 3M™ 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 10000 x g for 00:02:00 .
2m
Transfer 150 µL of supernatant to fresh low-binding tube and keep for second enrichment step, see .
Use residual supernatant to transfer beads to equilibrated C8 Stage tips.
Centrifuge for 00:02:00 at 500 x g and add flow-through to supernatant fraction from step .
2m
Wash beads that were retained by C8 Stage tips with 50 µL Wash buffer 1 (80 % (v/v) ACN, 1 % (v/v) TFA) and centrifuge at 500 x g for 00:02:00 .
2m
Wash beads with 50 µL Wash buffer 2 (10 % (v/v) ACN, 0.2 % (v/v) TFA) and centrifuge at 500 x g for 00:02:00 . Transfer tips to new vial.
2m
Prepare low-binding tubes with 60 µL 10 % (v/v) formic acid for phosphopeptide elutions to directly acidify them and minimize loss of phosphorlyation by ammonium hydroxide.
Elute bound phosphopeptides with 30 µL Elution buffer 1 (1 % (v/v) NH4OH) by centrifugation at 500 x g for 00:02:00 and directly transfer elution to prepared tubes ( ).
2m
Elute residual bound phosphopeptides with 30 µL Elution buffer 2 (5 % (v/v) NH4OH, 25 % (v/v) ACN) by centrifugation at 500 x g for 00:02:00 and directly transfer elution to prepared tubes ( ).
2m
Repeat phosphopeptide protocol with retained supernatant from and repeat protocol with Loading buffer 2 (20 % (v/v) Lactic acid solution, 85 %Merck MilliporeSigma (Sigma-Aldrich)Catalog #252476 , 70 % (v/v) ACN, 5 % (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.
Mass spectrometric analysis
6h 8m
Resuspend samples in mass spectrometric (MS) buffer (3 % (v/v) ACN, 0.1 % (v/v) formic acid), measure peptide concentration by Nanodrop and adjust concentration to 0.25 µg/µL if possible.
Load 500 ng peptides onto a 50 cm Acclaim™ PepMap™ 100 C18 HPLC ColumnsThermo ScientificCatalog #164945 connected to an EASY-nLC 1200 nano-LC system coupled to a QExactive HF mass spectrometer.
For normal peptides (Fraction 1), resolve peptides across a 03:38:00 active gradient at a 150 nL/min flow rate.
For phosphopeptides (Fraction 2), resolve peptides across a 02:30:00 min active gradient at a 150 nL/min flow rate.
6h 8m
Perform mass spectrometric analysis in data-independent acquisition mode with 24 (Fraction 1) or 20 (Fraction 2) variable windows with 1 m/z overlaps.
Record full mass spectra in the Orbitrap at a resolution of 120K in the range of 300-1650 m/z (Fraction 1) or 350-1600 m/z (Fraction 2) with a maximum injection time of 60 ms and an AGC target of 3e6.
Isolate precursor ions in the quadrupole and fragment them with stepped HCD at 28 ±3 % NCE (normalized collision energy).
Record fragment mass spectra in the Orbitrap at a resolution of 30K, an AGC target of 1e6 and maximum injection time set to auto.
Data analysis
Analyze raw data with Spectronaut in directDIA mode.
For Fraction 1, normal peptides, use default BGS settings except for minimal peptide lengths 6.
For Fraction 2, phosphopeptides, change default BGS settings to also include variable modifications phosphorylation at S, T, and Y, deamidation at N and Q as well as glutamine to pyro-glutamine modification. Set the Normalization Type Filter to include phosphorylated residues. Enable the PTM workflow and set the PTM localization filter to 0.75.
Use Swissprot database containing protein sequences from homo sapiens and additionally, a contaminant database like this one:
Citation
LINK
Retain only proteins or peptides that were confidently identified with a Q-value ≤ 0.01.
Export quantification results as pivot report for statistical analysis and hypothesis testing.
Use an appropriate tool for statistical analysis. For example, you can use Perseus.
Citation
LINK
For statistical analysis of Fraction 1:
Load data from Spectronaut into your tool.
Filter out Contaminants
Log2 transform quantities.
Group all replicates of one condition so you can use these groups for subsequent filtering and analysis.
Filter out proteins that have been identified and quantified inconsistently in less than 3 or 4 of 4 biological replicates across all conditions, i.e. if a protein has been identified sparsely in all conditions, filter it out, but if it is identified in 3 or 4 of 4 replicates in one condition or more, keep it.
Filter out proteins that have been identified and quantified based on only 1 precursor.
Impute missing values using a tail-based imputation. This step is crucial to perform hypothesis tests.
Use two-sample t-tests to determine significant differences between two conditions and multiple sample t-tests (ANOVA-based) to determine significant differences between more than 2 conditions.
Filter results for significantly changed proteins
You can also perform a post-hoc Tukey test based on the results from the ANOVA to determine between which conditions significant differences were identified. Then, use these results to filter out hits that are enriched in the control or filter hits that are significantly changed between certain conditions.
Normalize values to z-scores and average replicates of the same condition.
Perform hierarchical cluster analysis to identify and visualize cluster of protein that behave similarly and are enriched or depleted for one or more conditions.
Use clusters from previous step to perform further interactome analysis.
For statistical analysis of Fraction 2
Use the phosR package to process phosphosites.
Citation
LINK
Keep only phosphosites that were identified in more than 2 of 4 replicates for at least one condition.
Impute missing values first using a site- and condition specific imputation, and then a paired-tail imputation and normalize resulting values.
Export data from phosR and continue in your statistical tool of choice or continue in R.
Perform hypothesis tests for phosphosites as described for proteins above.
Protocol references
Tyanova, S. et al. (2016) ‘The Perseus computational platform for comprehensive
analysis of (prote)omics data’, Nature Methods. Nature Publishing Group,
pp. 731–740. Available at: https://doi.org/10.1038/nmeth.3901.
Kim, H.J. et al. (2021) ‘PhosR enables processing and functional analysis of
phosphoproteomic data’, Cell Reports, 34(8), p. 108771. Available at:
https://doi.org/10.1016/J.CELREP.2021.108771.
Bruderer, R. et al. (2017) ‘Optimization of Experimental Parameters in
Data-Independent Mass Spectrometry Significantly Increases Depth and
Reproducibility of Results’, Molecular & Cellular Proteomics,
16(12), p. 2296. Available at: https://doi.org/10.1074/MCP.RA117.000314.
Utriainen, M. and Morris, J.H. (2023) ‘clusterMaker2: a major update to clusterMaker, a
multi-algorithm clustering app for Cytoscape’, BMC Bioinformatics,
24(1), pp. 1–28. Available at:
https://doi.org/10.1186/S12859-023-05225-Z/FIGURES/10.
Citations
Step 51
Tyanova S, Cox J. Perseus: A Bioinformatics Platform for Integrative Analysis of Proteomics Data in Cancer Research.
https://doi.org/10.1007/978-1-4939-7493-1_7Step 53.1
Kim HJ, Kim T, Hoffman NJ, Xiao D, James DE, Humphrey SJ, Yang P. PhosR enables processing and functional analysis of phosphoproteomic data.
https://doi.org/10.1016/j.celrep.2021.108771Acknowledgements
This research was funded by Aligning Science
Across Parkinson’s (grant ASAP-000519 to S.R.P., S.K. and F.S) through the Michael J. Fox
Foundation for Parkinson’s Research (MJFF). This work was also supported by
funding of the German Research Foundation (DFG, F.S.: 496470458 and 516836828). We thank the laboratory of Samara Reck-Peterson for providing us with the initial protocol for BioID on which parts of this protocol are based.