TITLE: Cross-linking/MS-analysis of Thr72-phosphorylated Rab8A and PPM1H (D288A) complex
AUTHORS: Pawel Lis, Pui Yiu Lam, Axel Knebel , Kerryn Berndsen, Dario Alessi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A subset of Rab proteins, including Rab8A, have been identified as substrates of the Leucine Rich Repeat Kinase 2 (LRRK2) and the Protein Phosphatase PPM1H (Steger et al. 2017; Berndsen et al. 2019). As the crystal structure of the complex of PPM1H with Rab8A pT72 is unavailable, we employed a cross-linking approach (Iacobucci et al. 2018) to perform a study to identify the topology of this phosphatase-substrate binding. A ‘substrate-trapping’ mutant (D288A) of PPM1H was identified (Berndsen et al. 2019), and used in this study to form stable complex of PPM1H with Rab8A pT72. We then used Disuccinimidyl Dibutyric Urea (DSBU) to cross-link the complex of PPM1H (D288A) ‘substrate-trapping’ mutant and phospho-Rab8A. DSBU is a mass spectrometry cleavable amine reactive crosslinker with a 12.5 Å spacer arm that is widely used to identify and map sites of protein-protein interactions (Pan et al. 2018). This reagent cross-links Lys residues to acidic and hydroxyl amino acids located within 32 Å (Götze et al. 2019). Crosslinked samples were excised from a Coomassie-stained poly acrylamide gel and digested with 3 conditions (trypsin, trypsin/Asp-N and trypsin/Glu-C). In addition, SCX cartridge purification was applied in one of the tryptic digested samples to further enrich the cross-linked peptides. PPM1H and pRab8a cross-linked peptides were identified using meroX software (Götze et al. 2012). Potential crosslinked peptides with score higher than 50 as well as false discovery rate (FDR) less than 5% were manually inspected to confirm only a single crosslinked site was proposed from each peptide (Iacobucci et al. 2018).</div></div>

[STEPS]
?. [Protein buffer exchange]
Prepare a beaker containing  of  HEPES ,  NaCl,  MgCl2 buffer and a magnetic stir bar. Set the beaker on a magnetic stirrer with gentle stirring, at .
As the cross-linking reaction can be inhibited by amine containing buffers, a buffer exchange step for both, PPM1H and pRab8A protein solutions is needed prior to the crosslinking. Proteins should be dialysed into 40 mM HEPES pH 7.5, 150 mM NaCl, 2 mM MgCl2 buffer.
500 mL
0 Room temperature
?. [Protein buffer exchange]
Pipette up to  of each protein into separate Diacon dialysis tubes. Put the tubes in a floating rack, and place the rack on the surface of the buffer in the beaker. Incubate for  at .
0.5 mL
0 Room temperature
?. [Protein buffer exchange]
Transfer the floating rack with the dialysis tubes into a fresh beaker containing  of  HEPES ,  NaCl,  MgCl2 buffer and a magnetic stir bar. Set the beaker on a magnetic stirrer with gentle stirring, at . Incubate  at .
500 mL
0 Room temperature
0 Room temperature
?. [Preparation of the cross-linked pRab8A/PPM1H complex samples]
Prepare a solution of stoichiometrically Thr72 phosphorylated Rab8A at a concentration  (~, migrates at 20 kDa) and PPM1H at  (~, migrates at 50 kDa) in total volume of  using  HEPES ,  NaCl,  MgCl2 as a dilution buffer, to achieve 1.25-fold molar excess of Rab8A to PPM1H.
15.5 µl
The mixed volumes will depend on protein concentrations obtained after the buffer exchange step.
?. [Preparation of the cross-linked pRab8A/PPM1H complex samples]
Mix the protein solution gently and incubate at  for .
30 °C
?. [Preparation of the cross-linked pRab8A/PPM1H complex samples]
After this incubation incubate at  for .
30 Room temperature
?. [Preparation of the cross-linked pRab8A/PPM1H complex samples]
During this time prepare a fresh  stock solution of the cross-linker by dissolving  of DSBU in  of anhydrous DMSO, vortex briefly, keep , use within .
1 mg
15.5 µl
on ice
?. [Preparation of the cross-linked pRab8A/PPM1H complex samples]
Add  of the cross-linker solution into  of the protein solution, mix gently.
0.5 µl
15.5 µl
This will result in final concentration of   of DSBU (250-fold molar excess to pRab8A, 335-fold molar excess to PPM1H) in   of reaction mix.
?. [Preparation of the cross-linked pRab8A/PPM1H complex samples]
Incubate the cross-linking reaction for  at . Add  of  Tris  to quench the reaction, mix gently.
30 Room temperature
2 µl
?. [Preparation of the cross-linked pRab8A/PPM1H complex samples]
Add  of 4 x NuPAGE™ LDS Sample Buffer (containing lithium dodecyl sulfate at  with SERVA Blue G250 and phenol red), vortex. Immediately resolve the samples using SDS-PAGE.
7 µl
Do not add reducing agent such a 2-mercaptoethanol or heat the samples before electrophoresis as this could impact crosslinking reactions.
?. [SDS-PAGE and in-gel digestion]
Resolve the samples on a 4-12% gradient NuPAGE Bis-Tris gel. The final reaction volume is . Load  aliquots of each sample into 4 lanes (one for each protease condition: Sample A – trypsin, Sample B – trypsin+SCX, Sample C – trypsin+AspN, Sample D – trypsin+GluC). Run the gel at 70 V for , and then at 120 V until the dye front leaves the gel.
25 µl
6 µl
?. [SDS-PAGE and in-gel digestion]
Fix and stain the gel using Invitrogen™ Colloidal Blue Staining Kit
?. [SDS-PAGE and in-gel digestion]
Prepare the fixing solution (10% (v/v) glacial acetic acid in 50% (v/v) methanol) by mixing  of acetic acid with  of methanol and  of milliQ water.
5 mL
25 mL
20 mL
?. [SDS-PAGE and in-gel digestion]
Incubate the gel in the fixing solution for  at  with gentle shaking. Discard the fixing solution.
30 Room temperature
?. [SDS-PAGE and in-gel digestion]
Prepare the staining solution (20% (v/v) methanol, 20% (v/v) Stainer A, 5% (v/v) Stainer B). Mix  of Stainer A with  of methanol and  of milli-Q water. Do not add Stainer B at this stage. Incubate the gel in the solution for  at  with gentle shaking.
10 mL
10 mL
22.5 mL
30 Room temperature
?. [SDS-PAGE and in-gel digestion]
Add  of Stainer B directly to the solution containing the gel. Incubate the gel in the staining solution for  –  at  with gentle shaking.
2.5 mL
30 Room temperature
?. [SDS-PAGE and in-gel digestion]
Rinse the gel using milli-Q water at least 3 times within at least  or .
?. [SDS-PAGE and in-gel digestion]
Image the gel.
Note: Band-shifts corresponding to a PPM1H dimer size (100 kDa), and a tetramer of PPM1H and pRab8A (140 kDa) are an indication of a successful cross-linking (Figure 1). Decide which crosslinked species need to be excised. We excised the pRab8A-PPM1H crosslinked species and the PPM1H dimer crosslinked species highlighted on Figure 1.
?. [SDS-PAGE and in-gel digestion]
Prepare low-binding  Eppendorf tubes – one for each condition (A, B, C, D).
1.5 mL
?. [SDS-PAGE and in-gel digestion]
Using a fresh, sterile scalpel, excise each band of interest, cut into  cubes, and place the gel pieces in a.
1
?. [SDS-PAGE and in-gel digestion]
Add  of acetonitrile and incubate at  for  to dehydrate the gel pieces.
500 µl
30 Room temperature
?. [SDS-PAGE and in-gel digestion]
Discard the supernatant.
?. [SDS-PAGE and in-gel digestion]
Add  of freshly prepared  DTT solution and ensure gel pieces are covered. If necessary, add an additional  of  DTT to cover gel pieces.
50 µl
50 µl
?. [SDS-PAGE and in-gel digestion]
Incubate in a Thermomixer at  for  with mixing ().
56 °C
Centrifuge: 1200 33
?. [SDS-PAGE and in-gel digestion]
Add  of acetonitrile.
500 µl
?. [SDS-PAGE and in-gel digestion]
Incubate at  for  without shaking to dehydrate the gel pieces.
56 Room temperature
?. [SDS-PAGE and in-gel digestion]
Discard the supernatant.
?. [SDS-PAGE and in-gel digestion]
Prepare a fresh solution of  Iodoacetamide (IAA) in  ABC.
Please note that iodoacetamide is toxic and should be carefully weighed out in a fume hood (for details refer to the safety data sheet).
?. [SDS-PAGE and in-gel digestion]
Add - of freshly prepared  iodoacetic acid solution to ensure that gel pieces are covered.
50 µl
100 µl
?. [SDS-PAGE and in-gel digestion]
Cover reaction mixture from light by covering the tubes with aluminium foil.
?. [SDS-PAGE and in-gel digestion]
Incubate in a Thermomixer at  for  with mixing ().
56 Room temperature
Centrifuge: 1200 33
?. [SDS-PAGE and in-gel digestion]
After this incubation add  of acetonitrile.
500 µl
?. [SDS-PAGE and in-gel digestion]
Incubate at  for  to dehydrate the gel pieces without shaking.
56 Room temperature
?. [SDS-PAGE and in-gel digestion]
Discard the supernatant.
Please note that the solution contains toxic iodoacetamide and should be disposed of in an appropriate manner. For details refer to the safety data sheet.
?. [SDS-PAGE and in-gel digestion]
Add  of  ABC in 50% (v/v) acetonitrile.
100 µl
?. [SDS-PAGE and in-gel digestion]
Incubate at  for  without shaking.
56 Room temperature
?. [SDS-PAGE and in-gel digestion]
Add  of acetonitrile. Do not discard the supernatant from the previous step.
500 µl
?. [SDS-PAGE and in-gel digestion]
Incubate at  for  to dehydrate the gel pieces.
56 Room temperature
?. [SDS-PAGE and in-gel digestion]
Discard the supernatant.
?. [SDS-PAGE and in-gel digestion]
Prepare fresh  solution of  Trypsin in milli-Q water keep .
80 µl
on ice
?. [SDS-PAGE and in-gel digestion]
Prepare fresh  solution , AspN in milli-Q water keep .
20 µl
on ice
?. [SDS-PAGE and in-gel digestion]
Prepare fresh  solution , GluC in milli-Q water keep .
20 µl
on ice
?. [SDS-PAGE and in-gel digestion]
Use 4 different protease digestion conditions: Sample A – trypsin, Sample B – trypsin+SCX enrichment, Sample C – trypsin+AspN, Sample D – trypsin+GluC.
?. [SDS-PAGE and in-gel digestion]
For A add  of Trypsin solution to the dehydrated gel pieces. If the gel pieces are not covered completely, add  ABC solution to cover them.
20 µl
?. [SDS-PAGE and in-gel digestion]
For B add  of Trypsin solution to the dehydrated gel pieces. If the gel pieces are not covered completely, add  ABC solution to cover them.
20 µl
?. [SDS-PAGE and in-gel digestion]
For C add  of Trypsin solution to the dehydrated gel pieces, followed by  of AspN solution. If the gel pieces are not covered completely, add  ABC solution to cover them.
20 µl
20 µl
?. [SDS-PAGE and in-gel digestion]
For D add  of Trypsin solution to the dehydrated gel pieces, followed by  of GluC solution. If the gel pieces are not covered completely, add  ABC solution to cover them.
20 µl
20 µl
?. [SDS-PAGE and in-gel digestion]
Incubate all 4 samples  at  with mixing ().
30 °C
Centrifuge: 1200 33
?. [SDS-PAGE and in-gel digestion]
Prepare fresh peptide elution solution (1.67% (v/v) TFA in acetonitrile) by mixing 2 volumes of ACN with 1 volume of 5% (v/v) TFA solution.
?. [SDS-PAGE and in-gel digestion]
Add  peptide elution solution to each of the digestion tubes.
100 µl
?. [SDS-PAGE and in-gel digestion]
Incubate at  for  with mixing ().
37 °C
Centrifuge: 1200 33
?. [SDS-PAGE and in-gel digestion]
Transfer the supernatant that contains the digested protein into a fresh  low-binding Eppendorf tube. Avoid collecting any gel pieces.
1.5 mL
?. [SDS-PAGE and in-gel digestion]
In order to extract the remaining peptides present in the gel pieces, repeat the elution step by adding another  into the gel pieces.
100 µl
?. [SDS-PAGE and in-gel digestion]
Incubate at  for .
37 °C
?. [SDS-PAGE and in-gel digestion]
Collect the supernatant and combine with the previously collected supernatant in step 46.
?. [SDS-PAGE and in-gel digestion]
Freeze the samples on dry ice.
?. [SDS-PAGE and in-gel digestion]
Vacuum dry using SpeedVac.
?. [MCX SPE cartridge for cross-linked peptide enrichment]
Place the tube in a water bath sonicator at  for .
MCX cartridge contains mixed-mode sorbent (reversed-phase and cation exchange) which provides higher selectivity and sensitivity for cross-linked peptides enrichment than traditional SCX cartridge (Schmidt and Sinz 2017). Herein, we applied a similar protocol as previous study to enrich one of our tryptic digested samples (Iacobucci et al. 2018). Take tube labelled B from Freeze dried sample from step 51 and add    aqueous solution containing 4% (v/v) H3PO4.
37 Room temperature
?. [MCX SPE cartridge for cross-linked peptide enrichment]
Wash the MCX cartridge with  methanol (no syringe or manifold was applied; according to the nature protocol (Iacobucci et al. 2018), slow sample loading can enhance the interaction between the cross-linked peptides and cartridge). Wash the MCX cartridge with 2 aqueous solution containing 4% (v/v) H3PO4.
2 mL
?. [MCX SPE cartridge for cross-linked peptide enrichment]
Load sample from step 12 using a  pipette onto the washed MCX cartridge.
1000 µl
?. [MCX SPE cartridge for cross-linked peptide enrichment]
Wash the MCX cartridge with  aqueous solution containing 4% (v/v) H3PO4.
500 µl
?. [MCX SPE cartridge for cross-linked peptide enrichment]
Wash the MCX cartridge with  of 10% (v/v) MeOH solution with 0.1% (v/v) FA.
500 µl
?. [MCX SPE cartridge for cross-linked peptide enrichment]
Elute low-charged peptides by washing the MCX cartridge with  solution composed of  NH4OAc in 40% (v/v) MeOH solution with 0.1% (v/v) FA.
500 µl
?. [MCX SPE cartridge for cross-linked peptide enrichment]
Elute high-charged peptides by washing the MCX cartridge with  solution composed of  NH4OAc in 80% (v/v) MeOH solution with 0.1% (v/v) FA.
700 µl
?. [MCX SPE cartridge for cross-linked peptide enrichment]
Freeze the samples on dry ice.
?. [MCX SPE cartridge for cross-linked peptide enrichment]
Vacuum dry using SpeedVac
?. [LC MS/MS experiment]
Re-suspend all vacuum dried samples in  solution containing 3% (v/v) acetonitrile and 0.1% (v/v) FA.
Liquid chromatography tandem mass spectrometry (LC MS/MS) experiment was performed on an Ultrimate 3000 RSLC nano-HPLC system coupled to an Orbitrap ExplorisTM 480 mass spectrometer.
30 µl
?. [LC MS/MS experiment]
Place tube in a water bath sonicator at  for .
37 Room temperature
?. [LC MS/MS experiment]
Load –  solution from each sample onto the nano-HPLC system individually. Trap the peptides by a precolumn (Acclaim PepMapTM 100, C18, 100 µm x 2 cm, 5 µm, 100 Å) using aqueous solution containing 0.1% (v/v) TFA. Seperate the peptides by an analytical column (PepMapTM RSLC C18, 75 µm x 50 cm, 2 µm, 100 Å) at  using a linear gradient of 1 to 35% solvent B (solution containing 80% (v/v) acetonitrile and 0.1% (v/v) FA) for , 35 to 85% (v/v) solvent B for , 85% (v/v) solvent B for , 85 to 1% (v/v) solvent B for , and 1% (v/v) solvent B for . Set the flow rate at  for all experiments.
3 µl
14 µl
45 °C
300
?. [LC MS/MS experiment]
Acquire the data with data-dependent MS/MS mode. For each MS scan, set the scan range between 375 and 1500 m/z with the resolution at 120,000 and use 300% automatic gain control (AGC).
The maximum injection time for each MS scan was 100 ms. The 10 highest abundant peptides with charge state between 2 and 8 as well as intensity threshold higher than 1.0e+4 were then isolated with a 1.2 Da isolation window sequentially. Stepped HCD with normalized collision energy of 27, 30, and 33% was applied to fragment the isolated peptides. For each MS/MS scan, the resolution was set at 15,000 with a normalized AGC at 200%. The maximum injection time was set at 250 ms.Dynamic exclusion with 60 s duration and 2 ppm window was enabled for the experiment.
?. [Cross-linked peptide data analysis]
Convert the .RAW files obtained from the LC MS/MS experiments into .mgf files using RawConverter software (He et al. 2015).
?. [Cross-linked peptide data analysis]
Submit the .mgf files to search using MeroX software against PPM1H and Rab-8A protein sequences to identify potential cross-linked peptides (He et al. 2015).
Digestive enzyme, trypsin, trypsin and AspN, or trypsin and GluC, were selected according to the experimental setup. 3 maximum missed cleavages with peptide length ranged from 3 to 50 were applied. Carbamidomethylation at Cysteine residue was set as fixed modification; while oxidation at methionine residue and deamidation at asparagine residue were included in variable modification. DSBU cross-linker was selected with specificity cross-linked sites at Lysine, Serine, Threonine, and Tyrosine residues. 10 ppm and 20 ppm were used to filter the mass error in precursor ion (MS1) and fragment ion (MS2) scans. Only ions with signal-to-noise ratio high than 2 were used for database search. RISEUP searching mode was applied, minimum 2 fragments per peptide and 5% false discovery rate (FDR) were required for a cross-linked peptide identification.
?. [Cross-linked peptide data analysis]
Verify the potential cross-linked peptides with score higher than 50 to guarantee the cleavage information obtained from the MS experiment (Figure 2 and 3).