TITLE: Identification of PKC-regulated phosphosites on LRRK1 by mass spectrometry analysis
AUTHORS: Asad  Malik, Raja  Sekhar Nirujogi, Toan K. Phung, Dario R. Alessi

[DESCRIPTION]
We describe a non-radioactive, mass spectrometry-based assay that we deploy for identifying novel PKC-regulated sites on LRRK1 that are responsible for activation of its kinase activity.


[STEPS]
SECTION: Preparation of lipid vesicles for PKC activation
1. Clean a disposable glass culture tube by washing three times with 100% methanol. Allow to air-dry.
SECTION: Preparation of lipid vesicles for PKC activation
2. Pipette 0.5 µL of Diacylglycerol (stock concentration is 10 mg/mL) and 5 µL of Phosphatidylserine (stock concentration is 10 mg/mL) into the cleaned and dried glass tube.
 

SECTION: Preparation of lipid vesicles for PKC activation
3. Vacuum dry lipids using a SpeedVac system for 10 min. This should leave a visible, translucent lipid pellet.
 


SECTION: Preparation of lipid vesicles for PKC activation
4. Resuspend lipids from step 3 in 50 µL of 25 millimolar (mM) HEPES pH 7.4 , 50 millimolar (mM) KCl. Vortex gently until pellet is no longer visible.

SECTION: Kinase Reaction: Phosphorylation of LRRK1 by PKC
5. Prepare a primary “2X master mix” containing 50 millimolar (mM) HEPES pH 7.5, 100 millimolar (mM) KCl, 0.2% (v/v) 2‐Mercaptoethanol, 20 millimolar (mM) MgCl2, 2 millimolar (mM) ATP, 2 millimolar (mM) CaCl2, 200 μg/ml Phosphatidylserine and 20 μg/ml Diacylglycerol.
SECTION: Kinase Reaction: Phosphorylation of LRRK1 by PKC
6. For each reaction, add 15 µL of the primary “2X master mix” to a clean Eppendorf tube.
SECTION: Kinase Reaction: Phosphorylation of LRRK1 by PKC
7. Add 7.5 µL of 200 nanomolar (nM) LRRK1 wild type protein (final concentration is 50 nanomolar (nM)) to each reaction and allow equilibration on ice for 5 min.

SECTION: Kinase Reaction: Phosphorylation of LRRK1 by PKC
8.  Start the kinase reaction by adding 7.5 µL of 400 nanomolar (nM) PKC Alpha protein (final concentration is 100 nanomolar (nM)).
 

 

SECTION: Kinase Reaction: Phosphorylation of LRRK1 by PKC
9. Transfer the Eppendorf tubes to the thermo mixer set at 30 °C, 1000 rpm. Incubate for 45 min.




SECTION: Kinase Reaction: Phosphorylation of LRRK1 by PKC
10. Stop the kinase reaction by adding 10 µL of 4X LDS loading buffer to the reaction mix to a final concentration of 1X.


SECTION: Kinase Reaction: Phosphorylation of LRRK1 by PKC
11. Incubate the samples for 5 min at 70 °C on a heat block before proceeding to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) section.

SECTION: SDS-polyacrylamide gel electrophoresis (SDS-PAGE):
12. Load samples onto a NuPAGE 4–12% Bis–Tris Midi Gel (ThermoFisherScientific, Cat#WG1402BOX or Cat#WG1403BOX), alongside pre-stained molecular weight markers (ranging from 10 kDa to 250 kDa). Rinse wells carefully with running buffer before loading samples.
 

SECTION: SDS-polyacrylamide gel electrophoresis (SDS-PAGE):
13. Electrophorese samples at 130V with MOPS SDS running buffer for 120 min or until the blue dye runs off the gel.

SECTION: SDS-polyacrylamide gel electrophoresis (SDS-PAGE):
14. Place gel in a clean glass 15 cm dish and cover with 15 mL-20 mL of InstantBlue® Coomassie Protein stain. Incubate on see-saw rocker for 60 min at Room temperature.

SECTION: SDS-polyacrylamide gel electrophoresis (SDS-PAGE):
15. Replace the InstantBlue® Protein stain with double distilled water and allow to de-stain at Room temperature 2 min before proceeding with peptide digestion as described in Total Protein Digestion section.



SECTION: Total Protein Digestion
16. Using a clean scalpel, excise stained-bands corresponding to LRRK1 from gel and cut into approximately 1mm2 gel pieces.
SECTION: Total Protein Digestion
17. Transfer the gel pieces into a low-bind tube.
SECTION: Total Protein Digestion
18. De-stain gel pieces by repeated 10 min washes in 40% (v/v) ACN in 40 millimolar (mM) NH4HCO3.
 



SECTION: Total Protein Digestion
19. Reduce peptides by addition of 100 µL of 5 millimolar (mM) DTT in 40 millimolar (mM) NH4HCO3. Incubate on thermomixer at 56 °C for 30 min, 1200 rpm.

SECTION: Total Protein Digestion
20.  Remove the DTT solution and incubate gel pieces in 40% (v/v) ACN in 40 millimolar (mM) NH4HCO3 for 10 min at Room temperature?
 

SECTION: Total Protein Digestion
21. Alkylate peptides by addition of 20 millimolar (mM) iodoacetamide in 40 millimolar (mM) NH4HCO3 and incubate at Room temperature for 30 min, 1200 rpm.
 

SECTION: Total Protein Digestion
22. Dehydrate gel pieces by washing in 100% (v/v) ACN for 10 min.
 

SECTION: Total Protein Digestion
23. Remove supernatant using a pipette and vacuum dry gel pieces to remove any residual CAN.
SECTION: Total Protein Digestion
24. Add 100 ng of protease in 100 µL of appropriate buffer (See Table 1) to the gel pieces from step 23 and incubate 10 min on thermomixer at 37 °C, 1200 rpm. 
 


 

SECTION: Peptide extraction
25. Supplement samples from step 24 with 50 µL of extraction buffer (80% ACN in 0.2% Formic Acid) and incubate on thermomixer at  Room temperature for 10 min at 1200 rpm.
SECTION: Peptide extraction
26. Centrifuge samples for 1 min at 2000 x g to pellet the gel pieces and using a pipette carefully transfer the supernatant to a new low-binding? tube.
 

SECTION: Peptide extraction
27. Repeat step 25 until the gel pieces appear completely dried. Each time, transfer the supernatant into the same tube (from step 26).
SECTION: Peptide extraction
28. Vacuum dry the combined supernatants (containing the digested peptides) and proceed with C18 clean-up protocol (as described in C18 stage-tip protocol section).
SECTION: C18 stage-tip protocol:
29.  
 

 Prepare single layer of C18 stage-tip using 16-gauge syringe needle[FT(1].
 
 
SECTION: C18 stage-tip protocol:
30. Resuspend the vacuum dried peptides from step 28 in 80 µL of Solvent A1 (0.1% (by vol) TFA in MQ-H2O).
SECTION: C18 stage-tip protocol:
31. Add 80 µL of 100% (by vol) ACN to the C18 stage-tip from Step 29 and centrifuge at 2000 x g for 2 min at Room temperature. Discard flow through.
 

SECTION: C18 stage-tip protocol:
32. Add 80 µL Solvent A1 (0.1% (by vol) TFA (by vol) in MQ-H2O)) and centrifuge at 2000 x g for 2 min at Room temperature. Discard flow through. Repeat this step.
 

SECTION: C18 stage-tip protocol:
33. Load the acidified peptide digest from Step 30 to the C18 stage-tip from step 32 and centrifuge at 1500 x g for 5 min at Room temperature.
 

SECTION: C18 stage-tip protocol:
34. Reapply the flow through to the C18 stage-tip column and centrifuge at 1500 x g for 5 min at Room temperature.

SECTION: C18 stage-tip protocol:
35. Add 80 µL of Solvent A1 (0.1% (by vol) TFA v/v) in MQ-H2O)?) to the C18 stage-tip column and centrifuge at 2000 x g for 2 min at Room temperature. Discard flow through. Repeat again.

SECTION: C18 stage-tip protocol:
36. Place the C18 stage-tip from step 35 into a new 1.5 ml low binding tube. 
 

SECTION: C18 stage-tip protocol:
37. Elute peptides from the C18 stage-tip by adding 40 µL of Elution buffer (Solvent B1: 40% (by vol) acetonitrile in 0.1% (by vol) TFA) in MQ-H2O and centrifuge at 1500 x g for 2 min.

SECTION: C18 stage-tip protocol:
38. Repeat step 37.
Elute peptides from the C18 stage-tip by adding 40 µL of Elution buffer (Solvent B1: 40% (by vol) acetonitrile in 0.1% (by vol) TFA) in MQ-H2O and centrifuge at 1500 x g for 2 min.
SECTION: C18 stage-tip protocol:
39.  Immediately snap freeze the eluted peptides from step 38 on dry ice and vacuum dry.
SECTION: C18 stage-tip protocol:
40. Perform mass spectrometry analysis of the peptides as described in LC-MS/MS analysis section.
SECTION: LC-MS/MS analysis
41.  
Dissolve the peptides in LC-Buffer (3% ACN (v/v) in 0.1% Formic acid (v/v)).
 

 
SECTION: LC-MS/MS analysis
42. Take 200 ng of the peptide digest of LRRK2 in 5 µL or 10 µL in LC-buffer and prepare it for the Evotips loading. The Evo tips are a versatile disposable trap columns that enables <0.1% carry-over between samples.

SECTION: LC-MS/MS analysis
43. Prepare the Evotips as described in the Protocol in PMID: 33367571.
SECTION: LC-MS/MS analysis
44. Place the Evotips on EvoSep autosampler and used the 30 sample per day (30SPD) method to execute the LC method through Xcalibur interface that is inline with Orbitrap Exploris 240 mass spectrometer.
SECTION: LC-MS/MS analysis
45. EvoSep LC system injects and executes a partial elution of the sample from Evotip and loads onto the long storage loop in which the pre-formed gradient generated at the initial step. Following the loading the High-pressure pump pushes the sample into the analytical column (ReproSil-Pur C18, 1.9 µm beads by Dr Maisch. #EV1113). 

SECTION: LC-MS/MS analysis
46.  The following MS instrument method can be constructed for the High-resolution HCD fragmentation analysis:

 

SECTION: Data analysis
47. 
Transfer the raw data to search with Thermo Scientific Proteome Discoverer 2.4 Software suite that is integrated with Sequest-HT search algorithm.
 


SECTION: Data analysis
48. We recommend creating a custom protein sequence FASTA file rather than using the entire Uniprot Human or Mouse proteome FASTA file. For example: Copy the Human LRRK1 FASTA sequence and past it into a Notepad++ and save with LRRK1.FASTA .
 

SECTION: Data analysis
49. Import the LRRK1.FASTA sequence into the PD 2.4 software.
SECTION: Data analysis
50. Construct the Processing and Consensus workflows

 


 


SECTION: Data analysis
51. If the database search is to be done using MaxQuant then refer below settings


 

SECTION: Data analysis and Visualization
52.  
Manually verify the MS/MS spectrum and phosphorylation localization score within PD2.4.
 
SECTION: Data analysis and Visualization
53. Now export the filtered Phosphosites from modifications table for each of the sample/category
SECTION: Data analysis and Visualization
54. Use the below scripts for parsing and combining the data to generate a heatmap representation.
 

The script below would first read phosphosite mapping result, then map them on to the original protein amino acid sequence through combining PeptideGroups and ModificationSites result text file. The data would be filtered by probability greater or equal to 75 and grouped by the different tryptic digestion enzymes used. Only entries with the highest abundance values according to the unique motif, position and sample condition are kept. Then based on the sequence length, the data was divided into instances of 500 amino acid continuous span on the protein sequence. Each of these instances would be used to create a heatmap where the abundance of the peptide would be the heatmap color, the sample condition would be presented on the X-axis while the position of the phosphosites are represented in the Y-axis in ascending order.