TITLE: Sample preparation protocol for targeted mass spectrometry analysis of pRabs, total Rabs, LRRK2-pS910, pS935 and total LRRK2
AUTHORS: Raja S. Nirujogi, Dario Alessi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Leucine rich repeat kinase2 (LRRK2) is one of the most commonly implicated kinase and mutations that hyperactivate kinase activity is seen in familial Parkinson’s disease. LRRK2 phosphorylates a sub-set of small Rab GTPase proteins within their Switch-II motif. We recently described a multiplexed targeted mass spectrometry assay to identify and quantify both phosphorylated Rab proteins and well known LRRK2 phosphorylation sites such as LRRK2-pS910/pS935(PMID: 33367571). Here we describe a detailed protocol for the sample preparation of cells/tissues that are derived from LRRK2 mutation knock-in models. We outline a simple Triton-X 100 or SDS lysis that are straightforward to capture solubilized proteins amenable for both immunoblotting and mass spectrometry analysis. A stepwise protocol to buffer exchange Triton-X 100/SDS using S-Trap columns and on-column trypsin digestion. We also describe an optimized condition to conjugate required antibodies for the selective enrichment of pRabs and LRRK2 phosphopeptides and total LRRK2 peptide including the protocol for peptide immunoprecipitation and subsequent Stage tip-based desalting of the samples prior to targeted mass spectrometry analysis. We provide a cautionary step’s in each sections of the protocol for a reproducible sample preparation. We believe this protocol is an easy handout to study LRRK2 activity both from cells/tissues including samples from human neutrophils.</div></div>

[STEPS]
?. [Lysate preparation from mice tissues]
Snap freeze mouse tissue immediately after isolation in liquid nitrogen and store at .
-80 °C
?. [Lysate preparation from mice tissues]
Pulverize frozen tissue in liquid nitrogen to a fine powder and snap freeze immediately in 2ml tubes. This is done using cell crusher kit.
Maintain all Pulverizer units in liquid nitrogen including spatula. After use wash the pulveriser with a tap water and clean thoroughly several times with  .
?. [Lysate preparation from mice tissues]
Weigh pulverized tissue powder on a weighing balance.
5-7% of the wet weight of tissue should correspond to a protein amount of ~   to  / .
Keep samples on dry ice during and after weighing. Take a 1.5 ml tube check the weight on weighing balance and subsequently tare the weight. Now take small scoop of sample using pre-chilled fine spatula to weigh the sample
?. [Lysate preparation from mice tissues]
Immediately add  of .
[ weight of the tissue]
[SDS lysis buffer]
For e.g.   please add  .
?. [Lysate preparation from mice tissues]
Put samples on an orbital shaker in cold room at .
33
?. [Lysate preparation from mice tissues]
Boil samples at  for  and allow to cool.
95 °C
?. [Lysate preparation from mice tissues]
Sonicate samples using a Diagenode Bioruptor (at high energy for 10 cycles (/)).
[ON]
[OFF]
?. [Lysate preparation from mice tissues]
Clarify lysate by centrifuging at .
Centrifuge: 20800 33, 30 min, 4 10
?. [Lysate preparation from mice tissues]
Measure protein amount using the Bicinchoninic acid assay (BCA) method in triplicate at 1:10 and 1:20 dilutions-repeat analysis if readings are not close.
?. [Lysate preparation from mice tissues]
Take  for Mass spectrometry analysis.
[protein amount]
In case of mice brain tissues the minimum recommended lysate amount is   to identify and quantify endogenous pRab proteins and LRRK2 pS910/pS935 and total LRRK2 peptide.
?. [Lysate preparation from cells]
For cells such as A549 or HEK293 or Mouse embryonic fibroblasts (MEFs): Culture them in 6cm or 10cm dish to a ~70% confluency and complete the required treatments such as MLi2 treatment for about .
?. [Lysate preparation from cells]
Harvest cells by washing with 1X plain DMEM media without any serum and aspirate the media completely.
?. [Lysate preparation from cells]
Lyse cells using 1% Triton-X 100 lysis buffer. Add  to a 6cm dish or  to a 10cm dish.
150 µl
350 µl
?. [Lysate preparation from cells]
Scrape cells using cell scraper and collect the lysate using 1 ml non-autoclaved tips into a protein lo binding tubes.
?. [Lysate preparation from cells]
Keep cells  for  and centrifuge at .
on ice
Centrifuge: 18000 34, 20 min, 4 10
?. [Lysate preparation from cells]
Estimate protein amounts using BCA assay.
?. [Lysate preparation from cells]
Take  for Mass spectrometry analysis (cell lysate or tissue lysate).
[protein amount]
?. [SDS-clean up and S-Trap assisted trypsin digestion]
Aliquot  in 1.5 ml Protein lo-binding tubes.
[ protein amount]
?. [SDS-clean up and S-Trap assisted trypsin digestion]
Perform reduction by adding 1:10 dilution of a solution of 0.1 M TCEP dissolved in 300 mM TEABC to bring final concentration to .
[TCEP]
?. [SDS-clean up and S-Trap assisted trypsin digestion]
Incubate on a Thermomixer with a gentle agitation at .
60 33
?. [SDS-clean up and S-Trap assisted trypsin digestion]
Bring tubes to  and add a 1:10 dilution of freshly prepared  dissolved in water.
60 Room temperature
[iodoacetamide]
It is critical that the samples are at room temperature prior to addition of iodoacetamide.
?. [SDS-clean up and S-Trap assisted trypsin digestion]
Incubate in dark on a Thermomixer with a gentle agitation at .
33
?. [SDS-clean up and S-Trap assisted trypsin digestion]
Quench alkylation by addition of a 1:10 dilution of a solution of 0.1 M TCEP dissolved in 300 mM TEABC to bring final concentration to .If samples are not quenched after alkylation proceed immediately to the next steps below.
[TCEP]
At this stage sample can be stored in   freezer.
?. [SDS-clean up and S-Trap assisted trypsin digestion]
Incubate on a Thermomixer with a gentle agitation at .
33
?. [SDS-clean up and S-Trap assisted trypsin digestion]
Add SDS to a final concentration of  from .
[SDS stock]
The lysate is already in 2% (by mass) SDS, so supplement with a stock of 20% (by mass) SDS in order to bring the final SDS concentration to 5% (by mass). For lysates with 1% Triton-x 100, it is important to add final 5% (by mass) SDS to the samples to ensure complete denaturation and subsequent capturing of fine protein particles by S-Trap columns.
?. [SDS-clean up and S-Trap assisted trypsin digestion]
Add a 1:10 dilution of  into the sample to make a final concentration of ~.
[phosphoric acid]
[phosphoric acid]
Note for example,   to  .
?. [SDS-clean up and S-Trap assisted trypsin digestion]
Dilute the sample 7 times in S-Trap wash buffer ( in   v/v).For example, if sample volume is  then add ( in   v/v).
[methanol]
[TEAB pH 7.1]
50 µl
[S-Trap wash buffer]
[methanol]
[TEAB pH 7.1]
If the lysate volume is more than 50 µl, scale S-Trap wash buffer accordingly i.e. 7 times the volume of the lysate
?. [SDS-clean up and S-Trap assisted trypsin digestion]
Prepare an S-Trap mini column in a 2 ml tube.
?. [SDS-clean up and S-Trap assisted trypsin digestion]
Add the diluted protein mixture from step 27 to the column.
?. [SDS-clean up and S-Trap assisted trypsin digestion]
Centrifuge briefly at  to capture the protein particles.
Centrifuge: 1000 34, 1 min
All centrifugation steps should be at room temperature. If you observe any clogging then increase the centrifugation speed up to a maximum of 4,000g or increase the centrifugation time up to 2-4 minutes. . If the total volume of Lysate +S-Trap buffer mixture is >500 µl then add it to the column multiple times until the sample passes through the column and discard flow through
?. [SDS-clean up and S-Trap assisted trypsin digestion]
Wash column with  for a total of 4 times (spin  between washes):
[S-Trap buffer]
Centrifuge: 1000 34, 1 min
Note that the protein remains bound on the column and SDS and buffer components that effect trypsin digestion are removed.
?. [SDS-clean up and S-Trap assisted trypsin digestion]
Wash column with Spin  (1/4)
[S-Trap buffer]
Centrifuge: 1000 34, 1 min
?. [SDS-clean up and S-Trap assisted trypsin digestion]
Wash column with Spin  (2/4)
[S-Trap buffer]
Centrifuge: 1000 34, 1 min
?. [SDS-clean up and S-Trap assisted trypsin digestion]
Wash column with Spin  (3/4)
[S-Trap buffer]
Centrifuge: 1000 34, 1 min
?. [SDS-clean up and S-Trap assisted trypsin digestion]
Wash column with Spin  (4/4)
[S-Trap buffer]
Centrifuge: 1000 34, 1 min
?. [SDS-clean up and S-Trap assisted trypsin digestion]
Move the S-Trap column to a clean 2 ml tube for digestion.
?. [SDS-clean up and S-Trap assisted trypsin digestion]
Add  containing  dissolved in .
[freshly dissolved trypsin solution]
[trypsin]
[TEAB (1:20)]
Prepare and store Trypsin stocks in Milli-Q water at   of 50µl vials and store in   freezer.
?. [SDS-clean up and S-Trap assisted trypsin digestion]
Centrifuge briefly at .
Centrifuge: 200 34, 1 min
?. [SDS-clean up and S-Trap assisted trypsin digestion]
Collect flow through and reapply the trypsin solution back onto the column being careful to avoid air bubbles.
?. [SDS-clean up and S-Trap assisted trypsin digestion]
Cap the tubes and incubate at  without shaking for .
47 °C
Do not shake as this causes bubbles and damages the column.
?. [SDS-clean up and S-Trap assisted trypsin digestion]
Incubate for another  at .
47 Room temperature
?. [SDS-clean up and S-Trap assisted trypsin digestion]
Add  then spin to elute. Place the eluate in a new 1.5 ml Eppendorf tube termed “eluate tube”.
[ 50 mM TEAB]
?. [SDS-clean up and S-Trap assisted trypsin digestion]
Next, add  and spin to elute. Also add this eluate to the “eluate tube”.
[0.15% (by vol) Formic Acid]
?. [SDS-clean up and S-Trap assisted trypsin digestion]
Finally, add  and spin to elute. Also add this eluate to the “eluate tube”.
[50% (by vol) Acetonitrile in 0.15% (by vol) formic acid]
3 eluates should have been added to the eluate tube.
?. [SDS-clean up and S-Trap assisted trypsin digestion]
Take -, vacuum dry and inject on MS to verify the digestion efficiency.
1 µl
[combined eluate]
Analyse data with a 70 min gradient run on QE HF-X or Orbitrap Lumos mass spectrometer in a F1-T-FT-HCD mode. Search data with Proteome Discoverer 2.1 or 2.4 version. Determine the digestion efficiency by plotting number of missed cleavages. Zero missed cleavages should be >75% and single missed cleavages should be between 20-23%
?. [SDS-clean up and S-Trap assisted trypsin digestion]
Estimate peptide amounts using CBQCA assay kit and take equal amounts of peptide digest for each sample and Vacuum dry using SpeedVac.
Aliquot   separately for total Rab analysis.
?. [Pan-pRab, UDD1, UDD2 and 8G10 antibodies conjugation to Protein A/G beads]
Take required amount of Protein A/G beads and wash with a total of . Centrifuge at .(e.g. For 24 samples with 2 rounds of immunoprecipitations  is required. /sample for one round of immunoprecipitation).
[slurry volume + reminder is PBS wash volume (1X PBS buffer)]
Centrifuge: 2000 34, 3 min
[bead slurry]
[slurry]
Let the beads settle down for   before aspirating the supernatant.
?. [Pan-pRab, UDD1, UDD2 and 8G10 antibodies conjugation to Protein A/G beads]
Repeat wash steps with   two more times:
[1X PBS]
?. [Pan-pRab, UDD1, UDD2 and 8G10 antibodies conjugation to Protein A/G beads]
Wash beads with . (1/2)
[1X PBS]
?. [Pan-pRab, UDD1, UDD2 and 8G10 antibodies conjugation to Protein A/G beads]
Wash beads with . (2/2)
[1X PBS]
?. [Pan-pRab, UDD1, UDD2 and 8G10 antibodies conjugation to Protein A/G beads]
Incubate an equimolar concentration of Pan-pRab, UDD1, UDD2 and 8G10 antibodies to give a concentration of  of slurry. (Example, for 24 samples with two round of Immunoprecipitation, you require  +  +  +  in a total of  which will give 50ng/µl concentration).
[Pan-pRab]
[UDD1]
[UDD2]
[8G10]
[protein A/G slurry]
Due to the high binding capacity of Protein A/G beads all four antibodies can be simultaneously conjugated with 25 µg can be multiplexed to conjugate with  . For 24 samples 12 µg of each of the antibodies are required i.e. 0.5 µg per sample
?. [Pan-pRab, UDD1, UDD2 and 8G10 antibodies conjugation to Protein A/G beads]
Add extra 1X PBS to a final volume of .
1 mL
?. [Pan-pRab, UDD1, UDD2 and 8G10 antibodies conjugation to Protein A/G beads]
Incubate at  for  on an end-to-end rotator.
4 °C
?. [Pan-pRab, UDD1, UDD2 and 8G10 antibodies conjugation to Protein A/G beads]
Post incubation, centrifuge at  and collect the supernatant to be used as a flow-through fraction to confirm by SDS-PAGE that the antibodies are bound to the beads completely.
Centrifuge: 2000 34, 2 min, 4 10
?. [Pan-pRab, UDD1, UDD2 and 8G10 antibodies conjugation to Protein A/G beads]
Wash with  for a total of 3 times:
[1X PBS]
?. [Pan-pRab, UDD1, UDD2 and 8G10 antibodies conjugation to Protein A/G beads]
Wash with . (1/3)
[1X PBS]
?. [Pan-pRab, UDD1, UDD2 and 8G10 antibodies conjugation to Protein A/G beads]
Wash with . (2/3)
[1X PBS]
?. [Pan-pRab, UDD1, UDD2 and 8G10 antibodies conjugation to Protein A/G beads]
Wash with . (3/3)
[1X PBS]
?. [Pan-pRab, UDD1, UDD2 and 8G10 antibodies conjugation to Protein A/G beads]
Now add 1X PBS to a final volume of  to achieve an antibody concentration of . This mixture is stable at  for up to two months. For a longer storage add .
480 µl
[beads slurry]
4 °C
[sodium azide]
?. [Preparation and storage of heavy/light stable synthetic peptides]
Heavy/light synthetic peptides generally come in a lyophilized form at a net amount of .
Our lab has pRab1, pRab3, pRab8, pRab10, pRab35 and pRab43 and LRRK2 pS910 and pS935 and total LRRK2 heavy synthetic peptides.
?. [Preparation and storage of heavy/light stable synthetic peptides]
Dissolve peptides in . Leave tubes  for about  and then sonicate using a water bath sonicator for .
[reconstitution buffer]
on ice
Reconstitution buffer:   in   in HPLC-grade water to achieve 10 pmol/µl.
?. [Preparation and storage of heavy/light stable synthetic peptides]
Vortex and centrifuge tubes using benchtop microfuge for .
?. [Preparation and storage of heavy/light stable synthetic peptides]
Now prepare dilutions using reconstitution buffer as required (e.g. 10pmol/ µl, 1pmol/µl, 100 fmol/µl and 10fmol/µl.)From these stocks prepare an equimolar concentration of all six pRabs+3 LRRK2 peptides to get 1pmol/µl or 100fmol/µl or 10fmol/µl concentrations to be Spiked into the peptide mixture just before immunoprecipitation. Store these stocks in  freezer.
-80 °C
For example, to achieve 1000 fmol/µl of an equimolar concentration of 200 µl final volume, take 20 µl from each peptide (6 Rabs+3LRRK2 peptides) from a stock of 10 pmol/µl to a total volume of 160 µl and then add 40 µl of reconstitution buffer to achieve a final concentration of 1000fmol/µl in 200 µl volume.
?. [Preparation and storage of heavy/light stable synthetic peptides]
Maintain 10 fmol/µl as working stocks so that  should give  net amount in immunoprecipitation reaction.
10 µl
To avoid pipetting errors it is recommended to maintain the desired amount that will be spiked into the peptide mixture to be at a minimum of 10µl. e.g. For 100 fmol add 10µl of 10 fmol/µl stock. If you intend to Spike 50 fmol amount, then add 10 µl of 5fmol/µl stock
?. [Peptide immunoprecipitation]
Spin the lyophilized peptides from Step 42, at  to ensure everything settles down at the bottom of the tube. Add , keep samples on water bath sonication for . The pH should be around .
Centrifuge: 17000 34, 2 min
[1X IAP buffer]
Use pH strips that have a fine gradient scale from pH 1.0 to pH 10, to adjust the pH
?. [Peptide immunoprecipitation]
If the digest is still acidic, adjust the pH by adding . Centrifuge at high speed at  for  to remove any insoluble traces and transfer supernatant to new 1.5 ml Eppendorf tube.
[1 M Tris pH 12.0]
-80 Room temperature
?. [Peptide immunoprecipitation]
Keep samples . In the meantime, aliquot required amount of antibody slurry from the stock as prepared in Step 49.
on ice
For each peptide immunoprecipitation containing up to  -  we use   containing   +   +   +   antibody. Also, add antibody cocktail using nonautoclaved cut tips.
Due to low abundance of pRabs+LRRK2 pS910, pS935 in brain the recommended peptide amount to be used is >600 µg peptide digest derived from 1 to 1.5mg starting lysate.
?. [Peptide immunoprecipitation]
Add  amount of an equimolar heavy stable peptides derived from step 55.
We use an equimolar mixture of 10µl of 10fmol/µl stock to achieve 100 fmol amount containing six pRabs and LRRK2 pS910, pS9135 and unmodified peptide
?. [Peptide immunoprecipitation]
Incubate the mixture at  for  on an end-to-end rotator.
4 °C
?. [Peptide immunoprecipitation]
After the incubation, centrifuge at . Collect the flow through and transfer it to new protein lo-binding 1.5 ml Eppendorf tubes for second round of immunoprecipitation.
Centrifuge: 1500 34, 2 min, 4 10
?. [Peptide immunoprecipitation]
Keep beads on an ice-block  until the second round of immunoprecipitation is completed.
on ice
?. [Peptide immunoprecipitation]
To the flowthrough from the 1st round of immunoprecipitation (Step 60), add another  containing  +  +  +  antibody and continue with the second round of immunoprecipitation for another  at  on an end-to-end rotator.
[antibody slurry]
[ Pan-pRab]
[UDD1]
[UDD2]
[8G10]
4 °C
?. [Peptide immunoprecipitation]
Centrifuge at . Collect the flow through and transfer it to new 1.5 ml Eppendorf tubes.
Centrifuge: 1500 34, 2 min, 4 10
This flow through can also be used for subsequent enrichment of a different analyte or second round of IP or phosphopeptides using TiO2.
?. [Peptide immunoprecipitation]
Add  to the beads and carefully transfer it to the beads that are kept  from 1st round of immunoprecipitation in step 61.
[1X IAP buffer]
on ice
?. [Peptide immunoprecipitation]
Add another  to the beads and gently wash tubes by inverting up-and-down for about 20 times.
[1X IAP buffer]
This can be easily done by keeping samples on an ice-block and cover it with silver foil. Now complete wash by inverting up-and-down steps for all tubes at once. Use this technique for all subsequent wash steps.
?. [Peptide immunoprecipitation]
Centrifuge tubes at . Place tubes on ice block and allow the beads to settle for at least  before aspirating the supernatant.
Centrifuge: 1500 34, 3 min, 4 10
?. [Peptide immunoprecipitation]
Repeat washing by adding  and centrifuge tubes at . Place tubes on ice block and allow the beads to settle for at least  before aspirating the supernatant.
[ice cold 1X IAP buffer]
Centrifuge: 1500 34, 3 min, 4 10
?. [Peptide immunoprecipitation]
Wash the beads with , Centrifuge tubes at . Place tubes on ice block and allow the beads to settle for at least  before aspirating the supernatant. (1/2)
[ice-cold 1X PBS]
Centrifuge: 1500 34, 3 min, 4 10
?. [Peptide immunoprecipitation]
Wash the beads with , Centrifuge tubes at . Place tubes on ice block and allow the beads to settle for at least  before aspirating the supernatant. (2/2)
[ice-cold 1X PBS]
Centrifuge: 1500 34, 3 min, 4 10
?. [Peptide immunoprecipitation]
Wash the beads with , Centrifuge tubes at . Place tubes on ice block and allow the beads to settle for at least  before aspirating the supernatant.
[ice-cold Milli-Q water]
Centrifuge: 1500 34, 3 min, 4 10
?. [Peptide immunoprecipitation]
After the final wash, centrifuge and aspirate all of the Milli-Q water and add  to the beads. Gently tap for proper mixing and incubate on a Thermo mixer at .
[0.2% (by vol) TFA]
33
?. [Peptide immunoprecipitation]
Centrifuge at  and collect the eluate into a new collection tube with a gel loader tip.
Centrifuge: 1500 34, 2 min
?. [Peptide immunoprecipitation]
Repeat the elution step by adding another  followed by incubation at  for  and centrifuge and collect the supernatant into the previous collection tube and proceed with C18 stage-tip clean up.
[0.2% (by vol) TFA]
4 Room temperature
?. [C18 Stage-tip]
Prepare single layer of C18 stage-tip using 16-gauge syringe needle as shown in figure 2.
?. [C18 Stage-tip]
Add  to the C18 stage-tip.
[100% (by vol) Acetonitrile]
Note this is required to activate the C18 resin.
?. [C18 Stage-tip]
Centrifuge at . Discard flow through.
Centrifuge: 2000 34, 2 min
?. [C18 Stage-tip]
Add .
[Solvent-A1 0.1% (by vol) TFA (by vol)]
Note this is required to equilibrate the C18 resin.
?. [C18 Stage-tip]
Centrifuge at . Discard flow through. (1/2)
Centrifuge: 2000 34, 2 min
?. [C18 Stage-tip]
Centrifuge at . Discard flow through. (2/2)
Centrifuge: 2000 34, 2 min
?. [C18 Stage-tip]
Load the acidified peptide digest from step 72 to Stage-tip.
?. [C18 Stage-tip]
Centrifuge at .
Centrifuge: 1500 34, 5 min
?. [C18 Stage-tip]
Reapply the flow through to the C18 stage-tip column and repeat this step. (1/2)
?. [C18 Stage-tip]
Reapply the flow through to the C18 stage-tip column. (2/2)
?. [C18 Stage-tip]
Add , and centrifuge at . Discard flow through. Repeat again. (1/2)
[solvent-A1 (0.1% (by vol) TFA v/v)]
Centrifuge: 2000 34, 2 min
?. [C18 Stage-tip]
Add , and centrifuge at . Discard flow through. (2/2)
[solvent-A1 (0.1% (by vol) TFA v/v)]
Centrifuge: 2000 34, 2 min
?. [C18 Stage-tip]
Elute peptides absorbed to C18 column by placing the stage-tips into new 1.5 ml low binding tubes.
?. [C18 Stage-tip]
Add .
[Elution buffer (Solvent B1- 50% (by vol) acetonitrile in 0.1% (by vol) TFA)]
?. [C18 Stage-tip]
Centrifuge at . Add another  and repeat the centrifugation at .
Centrifuge: 1500 34, 2 min
[Elution buffer (Solvent B1- 50% (by vol) acetonitrile in 0.1% (by vol) TFA)]
Centrifuge: 1500 34, 2 min
?. [C18 Stage-tip]
Immediately snap freeze on dry ice the eluates and vacuum dry the samples using a Speed-Vac.
?. [C18 Stage-tip]
Store samples in  freezer until the LC-MS/MS analysis.
-80 °C