GFP Immunoprecipitation and Sample Preparation for Tandem Mass Tag (TMT) Mass Spectrometry Analysis

Plate cells in 10 cm dishes (one dish for each experimental condition) to give a 60-70% confluency the following day (around 2.2 x 10⁶cells seeded per 10 cm dish).

Prepare a transfection mix in a sterile 1.5ml Eppendorf tube, containing (for each 10 cm dish):

A	B
FLAG-LRRK2 [Y1699C] plasmid	3 µg
wild type RILPL1-GFP or 2 µg [R293A] RILPL1-GFP plasmid	2 µg
HA-Rab8A [Q67L] plasmid	1 µg
1 mg/ml PEI Max 40K	18 µl
OptiMem	500 µl

Mix by vortexing and incubate at Room temperature for 0h 20m 0s.

Add the mixture dropwise to the cells from step 1 using a P1000 sterile pipette.

Incubate cells at 37°C for 24h 0m 0s.

Remove culture medium completely from each dish using an aspirator.

Quickly rinse cells in the tissue culture dish by carefully pouring culture media without Foetal bovine serum (at Room temperature) into the dish.

Pour off media from the culture dish and completely aspirate any residual media.

Immediately add 400µL of ice-cold complete lysis buffer to each dish ensuring that the entire surface is covered by lysis buffer.

Transfer the plate On ice.

Scrape the cells on the dish using a cell lifter to ensure all cells are detached from the dish.

Using a pipette, transfer the lysate to a 1.5mL Eppendorf tube.

Leave samples On ice for 20-30 minutes to allow for efficient lysis.

Spin down lysates at 17000x g,4°C.

Transfer supernatant to a new Eppendorf tube and discard the pellet.

Proceed to estimating the protein concentration of cell lysates by Bradford assay according to the manufacturer’s instructions.

Transfer n x 20µL of packed ChromoTek GFP-Trap Agarose Beads (where n = number of samples) into a low binding Eppendorf tube.

Pellet the beads by centrifuging at 2500x g,4°C.

Carefully aspirate the supernatant.

Resuspend the beads in 1mL of IP wash buffer.

Repeat steps 18 to 20 twice.

Centrifuge at 2500x g,4°C and aspirate the supernatant.

Resuspend beads from step 22 in n x 20µL of IP wash buffer (where n = number of samples) to make a 1:1 slurry.

Aliquot the washed beads from step 23 into fresh low-binding Eppendorf tubes (40µL of slurry for each sample, corresponding to 20µL of packed ChromoTek GFP-Trap Agarose Beads). Leave the tubes On ice until use.

For each sample, transfer 500µg lysate from step 15 to the washed beads.

Incubate for 2h 0m 0s at 4°C under mild agitation (on an orbital shaker).

Pellet the beads by centrifuging at 2500x g,4°C.

Carefully aspirate the supernatant.

Resuspend the beads in 1mL of IP wash buffer.

Repeat steps 27 to 29 twice.

Centrifuge at 2500x g,4°C and aspirate the supernatant.

Immediately proceed to Elution and on-bead tryptic digestion of immunoprecipitated proteins .

Add 100µL of elution buffer I to the beads from step 32.

Add 500ng of sequencing grade trypsin to the mixture and incubate on a Thermomixer at 800rpm.

Centrifuge the mixture at 2500x g. Carefully transfer the supernatant to new Eppendorf tubes, being careful not to disturb the beads.

Add 100µL of elution buffer II to the beads from step 35 and mix gently by tapping. Centrifuge the mixture at 2500x g. Carefully transfer the supernatant to the collection Eppendorf tubes from step 35, being careful not to disturb the beads.

Incubate the Eppendorf tubes on the Thermomixer 800rpm 2h 0m 0s (or minimum of 12 hr).

Add 1% (v/v) Trifluoroacetic Acid (TFA) to the digested peptides from step 37. Incubate for 0h 5m 0s at Room temperature and centrifuge at 17000x g.

Prepare a C18 stage-tip for each sample as described in dx.doi.org/10.17504/protocols.io.bs3tngnn .

C18 stage tips activation : add 80µL of 100% ACN to each stage-tip and centrifuge at 2000x g.

C18 stage tips equilibration : add 80µL of 0.1% TFA to each stage-tip and centrifuge at 2000x g.

Repeat step 41.

Transfer the C18 stage-tip to a new low-binding Eppendorf.

Load the digested peptides from step 38 onto the C18 stage-tip from step 43 and centrifuge at 2000x g.

Collect the flowthrough from step 44 and re-load onto the same C18 stage-tip. Centrifuge at 2000x g.

Wash the C18 stage-tips by adding 80µL of 0.1% TFA and centrifuging at 2000x g.

Repeat step 46.

Transfer the C18 stage-tip to a new low-binding Eppendorf.

Add 30µL of 30% (v/v) ACN in 0.1% (v/v) TFA to each stage-tip and centrifuge at 1000x g.

Repeat step 49.

Take 1-2µL of the digested peptides, vacuum dry and inject on MS to verify the digestion efficiency.

Vacuum dry completely the remaining peptides and store at -80°C until ready to undertake TMT labelling.

Dissolve 800µg of each of the TMT mass tag reagents within the 11-plex TMT reagent kit in 40µL of 100% anhydrous acetonitrile to obtain a 20μg/μL concentration for each TMT reporter tag.

Leave at Room temperature for 0h 10m 0s, then vortex and spin 2000x g.

Dissolve lyophilized peptides from step 52 in 50µL of a mixture containing 38µL 50millimolar (mM) TEAB buffer + 8µL 100% (by vol) anhydrous acetonitrile.

Place the samples in a water bath sonicator for 0h 10m 0s.

Centrifuge the samples 17000x g.

Transfer dissolved peptides into a 1.5ml protein low binding Eppendorf tube.

Add 10µL 20μg/μL TMT reagent i.e. 200µg aiming for a 1:1 mass ratio of peptide: TMT reagent.

Give a gentle vortex and spin at 2000x g.

Place samples on a Thermomixer and incubate with gentle agitation at 800rpm .

Add another 50µL 50millimolar (mM) TEAB buffer to make a final 100µL reaction. Vortex, brief spin at 2000x g and incubate on a Thermomixer for 0h 10m 0s.

In order to verify the TMT labelling efficiency of each TMT mass tag, take a 5µL aliquot from each of the TMT-labelled samples and pool the aliquots in a single tube. Vacuum dry immediately using a SpeedVac.

Store the remaining 95µL at -80°C until the labelling efficiency has been verified.

If the labelling efficiency is >98% and levels of each labelled peptide appear to be close to 1:1, then proceed with the below steps.

Thaw stored TMT labelled samples from step 63 to Room temperature.

Prepare 5% (by vol) final Hydroxyl amine solution by dissolving in water from a 50% (by vol) stock solution.

Add 5µL 5% (by vol) Hydroxylamine to each sample to quench TMT reaction by incubating the reaction at Room temperature on a Thermomixer for 0h 20m 0s.

Pool all samples into a single tube.

Transfer 20% of the reaction to a new low-binding Eppendorf tube as a backup: snap freeze on dry ice and vacuum dry.

Snap freeze the remaining 80% of the reaction and vacuum dry using Speed vac.

Prepare four C18 stage-tips as described in dx.doi.org/10.17504/protocols.io.bs3tngnn .

Label eight 1.5ml low-binding Eppendorf tubes as "Fraction 1" to "Fraction 8".

Prepare 50mL of bRP stock solution (50millimolar (mM) Ammonium formate in Milli-Q H₂O).

Prepare Solvent A : Mix 20mL of bRP stock solution with 20mL of Milli-Q H₂O (=25millimolar (mM) Ammonium formate in Milli-Q H₂O).

Prepare Solvent B : Mix 20mL of bRP stock solution with 20mL of 100% Acetonitrile (=25millimolar (mM) Ammonium formate in 50% ACN).

Prepare elution solvents for fractionation (required in steps 92 and 93) as described in the table below.

Prepare each elution solvent in a 2 ml Eppendorf tube.

A	B	C	D	E
Elution solvent # (Fraction number)	Final ACN % in Elution solvent		Solvent A (ml)	Total volume (ml)
8	100%	100% ACN	0	N/A
7	17.5%	0.7 ml Solvent B (50% ACN)	1.3	2.0
6	15.0%	1.2 ml Elution solvent 7 (17.5% ACN)	0.2	1.4
5	12.5%	1.0 ml Elution solvent 6 (15.0% ACN)	0.2	1.2
4	10.0%	0.8 ml Elution solvent 5 (12.5% ACN)	0.2	1.0
3	7.5%	0.6 ml Elution solvent 4 (10.0% ACN)	0.2	0.8
2	5.0%	0.4 ml Elution solvent 3 (7.5% ACN)	0.2	0.6
1	2.5%	0.2 ml Elution solvent 2 (5.0% ACN)	0.2	0.4

Dissolve peptides from step 58 in 200µL of Solvent A.

Place samples on a Thermomixer at 1800rpm.

Centrifuge sample at 17000x g.

Transfer the supernatant into a new 1.5ml protein low-binding Eppendorf tube.

Add 200µL of 100% ACN to the C18 stage-tips (Step 71) and centrifuge at 2500x g to activate the columns. Discard the flow through.

Add 200µL of Solvent B to each column from step 81 and centrifuge at 2500x g. Discard the flow through.

Add 200µL of Solvent A to each column from step 82 and centrifuge at 2500x g .

Transfer the column to a new low-binding Eppendorf tube.

Sample loading : Slowly load each sample (from step 80) onto a column (from step 84).

Centrifuge at 1500x g.

Collect the flowthrough from step 86 and slowly load onto the same column.

Centrifuge at 1500x g.

Transfer the column into a new 1.5ml Eppendorf tube.

To wash the column, add 200µL of Solvent A to the column and centrifuge at 2500x g.

Transfer the column into the tube labelled as “Fraction 1” (from step 72).

Add 60µL of Elution solvent 1 (from step 76) to the column and centrifuge at 1500x g.

Repeat steps 91 and 92 to generate Fraction 2 to Fraction 8. For each fraction, add 60 µl of the corresponding Elution solvent (from step 76) to the column and centrifuge at 1500x g.

Pool the 8 fractions from steps 92 and 93 as follows (to generate 4 final fractions):

• Pool fraction 1 and 5;
• Pool fraction 2 and 6;
• Pool fraction 3 and 7;
• Pool fraction 4 and 8.

Place fractions on dry ice and vacuum dry completely using a SpeedVac.

Dissolve each fraction from step 95 in 20µL of LC-buffer (3% (v/v) ACN, 0.1% (v/v) formic acid).

Place samples on a Thermomixer at 1800rpm.

Transfer 10µL of the sample from step 97 into a LC-vial for analysis (Step 99). The remaining sample can be stored at -80°C as a back-up.

Perform LC-MS/MS analysis on an Orbitrap Lumos Tribrid mass spectrometer in MS3 mode. The mass spectrometer instrument settings in data acquisition are described in the table below.

A	B
Application Mode	Peptide
Method Duration (min)	140
Global Parameters
Infusion Mode	Liquid Chromatography
Expected LC Peak Width (s)	30
Advanced Peak Determination	False
Default Charge State	2
Internal Mass Calibration	Off
Experiment#1 [MS]
Start Time (min)	0
End Time (min)	140
Master Scan
MS OT
Detector Type	Orbitrap
Orbitrap Resolution	120000
Mass Range	Normal
Use Quadrupole Isolation	True
Scan Range (m/z)	350-1500
RF Lens (%)	30
AGC Target	Custom
Normalized AGC Target (%)	50
Maximum Injection Time Mode	Custom
Maximum Injection Time (ms)	50
Micro scans	1
Data Type	Profile
Polarity	Positive
Source Fragmentation	Disabled
Scan Description
Filters
MIPS
Monoisotopic Peak Determination	Peptide
Charge State
Include charge state(s)	2-7
Include undetermined charge states	False
Dynamic Exclusion
Use Common Settings	False
Exclude after n times	1
Exclusion duration (s)	45
Mass Tolerance	ppm
Low	10
High	10
Exclude Isotopes	True
Perform dependent scan on single charge state per precursor only	True
Intensity
Filter Type	Intensity Threshold
Intensity Threshold	5.00E+03
Data Dependent
Data Dependent Mode	Number of Scans
Number of Dependent Scans	10
Scan Event Type 1
Scan
ddMS² OT HCD
Isolation Mode	Quadrupole
Isolation Window (m/z)	0.7
Isolation Offset	Off
Activation Type	HCD
Collision Energy Mode	Fixed
HCD Collision Energy (%)	39
Detector Type	Orbitrap
Orbitrap Resolution	30000
Mass Range	Normal
Scan Range Mode	Auto
AGC Target	Standard
Maximum Injection Time Mode	Custom
Maximum Injection Time (ms)	96
Micro scans	1
Data Type	Centroid
Use EASY-IC™	False
Scan Description
Filters
Precursor Selection Range
Selection Range Mode	Mass Range
Mass Range (m/z)	400-1200
Precursor Ion Exclusion
Exclusion mass width	ppm
Low	25
High	25
Isobaric Tag Loss Exclusion
Reagent	TMT
Data Dependent
Data Dependent Mode	Scans Per Outcome
Scan Event Type 1
Scan
ddMS3 OT HCD
MSⁿ Level	3
Synchronous Precursor Selection	True
Number of SPS Precursors	5
MS Isolation Window (m/z)	2
MS2 Isolation Window (m/z)	2
Isolation Offset	Off
Activation Type	HCD
HCD Collision Energy (%)	65
Detector Type	Orbitrap
Orbitrap Resolution	50000
Mass Range	Normal
Scan Range Mode	Define m/z range
Scan Range (m/z)	100-500
AGC Target	Custom
Normalized AGC Target (%)	200
Maximum Injection Time Mode	Custom
Maximum Injection Time (ms)	120
Micro scans	1
Data Type	Profile
Use EASY-IC™	False
Scan Description
Number of Dependent Scans	5

The raw data was searched using MaxQuant version 1.6.6.0 using the parameters described below.

A	B
Parameter	Value
Version	1.6.6.0
User name	Rnirujogi
Machine name	SILAC-MRC0
Date of writing	10/23/2019 21:11:33
Include contaminants	TRUE
PSM FDR	0.01
PSM FDR Crosslink	0.01
Protein FDR	0.01
Site FDR	0.01
Use Normalized Ratios For Occupancy	TRUE
Min. peptide Length	7
Min. score for unmodified peptides	0
Min. score for modified peptides	40
Min. delta score for unmodified peptides	0
Min. delta score for modified peptides	6
Min. unique peptides	0
Min. razor peptides	1
Min. peptides	1
Use only unmodified peptides and	TRUE
Modifications included in protein quantification	Oxidation (M);Acetyl (Protein N-term);Deamidation (NQ)
Peptides used for protein quantification	Razor
Discard unmodified counterpart peptides	TRUE
Label min. ratio count	1
Use delta score	FALSE
iBAQ	TRUE
iBAQ log fit	TRUE
Match between runs	TRUE
Matching time window [min]	0.7
Match ion mobility window [indices]	0.05
Alignment time window [min]	20
Alignment ion mobility window [indices]	1
Find dependent peptides	FALSE
Fasta file	D:\Database\HUMAN-Uniprot-150317_Custom7.FASTA
Decoy mode	revert
Include contaminants	TRUE
Advanced ratios	TRUE
Fixed andromeda index folder
Temporary folder
Combined folder location
Second peptides	FALSE
Stabilize large LFQ ratios	FALSE
Separate LFQ in parameter groups	FALSE
Require MS/MS for LFQ comparisons	FALSE
Calculate peak properties	FALSE
Main search max. combinations	200
Advanced site intensities	FALSE
Write msScans table	TRUE
Write msmsScans table	TRUE
Write ms3Scans table	TRUE
Write allPeptides table	TRUE
Write mzRange table	TRUE
Write pasefMsmsScans table	TRUE
Write accumulatedPasefMsmsScans table	TRUE
Max. peptide mass [Da]	4600
Min. peptide length for unspecific search	8
Max. peptide length for unspecific search	25
Razor protein FDR	TRUE
Disable MD5	FALSE
Max mods in site table	3
Match unidentified features	FALSE
Epsilon score for mutations
Evaluate variant peptides separately	TRUE
Variation mode	None
MS/MS tol. (FTMS)	20 ppm
Top MS/MS peaks per Da interval. (FTMS)	12
Da interval. (FTMS)	100
MS/MS deisotoping (FTMS)	TRUE
MS/MS deisotoping tolerance (FTMS)	7
MS/MS deisotoping tolerance unit (FTMS)	ppm
MS/MS higher charges (FTMS)	TRUE
MS/MS water loss (FTMS)	TRUE
MS/MS ammonia loss (FTMS)	TRUE
MS/MS dependent losses (FTMS)	TRUE
MS/MS recalibration (FTMS)	FALSE
MS/MS tol. (ITMS)	0.5 Da
Top MS/MS peaks per Da interval. (ITMS)	8
Da interval. (ITMS)	100
MS/MS deisotoping (ITMS)	FALSE
MS/MS deisotoping tolerance (ITMS)	0.15
MS/MS deisotoping tolerance unit (ITMS)	Da
MS/MS higher charges (ITMS)	TRUE
MS/MS water loss (ITMS)	TRUE
MS/MS ammonia loss (ITMS)	TRUE
MS/MS dependent losses (ITMS)	TRUE
MS/MS recalibration (ITMS)	FALSE
MS/MS tol. (TOF)	40 ppm
Top MS/MS peaks per Da interval. (TOF)	10
Da interval. (TOF)	100
MS/MS deisotoping (TOF)	TRUE
MS/MS deisotoping tolerance (TOF)	0.01
MS/MS deisotoping tolerance unit (TOF)	Da
MS/MS higher charges (TOF)	TRUE
MS/MS water loss (TOF)	TRUE
MS/MS ammonia loss (TOF)	TRUE
MS/MS dependent losses (TOF)	TRUE
MS/MS recalibration (TOF)	FALSE
MS/MS tol. (Unknown)	0.5 Da
Top MS/MS peaks per Da interval. (Unknown)	8
Da interval. (Unknown)	100
MS/MS deisotoping (Unknown)	FALSE
MS/MS deisotoping tolerance (Unknown)	0.15
MS/MS deisotoping tolerance unit (Unknown)	Da
MS/MS higher charges (Unknown)	TRUE
MS/MS water loss (Unknown)	TRUE
MS/MS ammonia loss (Unknown)	TRUE
MS/MS dependent losses (Unknown)	TRUE
MS/MS recalibration (Unknown)	FALSE
Site tables	Deamidation (NQ)Sites.txt;Oxidation (M)Sites.txt;Phospho (STY)Sites.txt