May 10, 2023
GFP Immunoprecipitation and Sample Preparation for Tandem Mass Tag (TMT) Mass Spectrometry Analysis
- Prosenjit Pal1,2,
- Raja S. Nirujogi1,2,
- Francesca Tonelli1,2,
- Dario R Alessi1,2
- 1MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, DD1 5EH, UK;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
Protocol Citation: Prosenjit Pal, Raja S. Nirujogi, Francesca Tonelli, Dario R Alessi 2023. GFP Immunoprecipitation and Sample Preparation for Tandem Mass Tag (TMT) Mass Spectrometry Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly7kxqlx9/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's working
Created: May 05, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 81447
Keywords: Immunoprecipitation of GFP-tagged proteins from cell lysates, Tandem Mass Tag (TMT) Mass Spectrometry Analysis, ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: ASAP-000463
Abstract
We describe a method to identify potential interactors of any Green Fluorescent Protein (GFP) tagged protein expressed in mammalian cells by GFP immunoprecipitation coupled to Tandem Mass Tag (TMT) mass spectrometry analysis. As an example, we used a GFP-tagged phosphoRab interactor protein (RILPL1-GFP), and its non-binding mutant (RILPL1 [R293A]-GFP, which cannot interact with phosphorylated Rab proteins) as a control.
Attachments
710-1531.docx
50KB
Guidelines
Protocol overview:
- Transient transfection of HEK293 cells for expression of GFP-tagged proteins.
- Preparation and quantification of cell lysates from HEK293 cells.
- Immunoprecipitation of GFP-tagged proteins from cell lysates.
- On-bead tryptic digestion and TMT labelling of immunoprecipitated proteins for LC-MS/MS mass spectrometry analysis.
Materials
Reagents
A. For cell culture, transient transfection and GFP immunoprecipitation:
- HEK293ATCCCatalog #CRL-1573 cultured in complete growth medium.
- Growth medium: Dulbecco’s Modified Eagle’s Medium (DMEM), High Glucose, no glutamine (GibcoTM, catalog number: 11960044, or equivalent) supplemented with 10% (v/v) Foetal Bovine Serum (FBS) (Sigma #F7524, or equivalent), 2 mM L-glutamine (GibcoTM, catalog number: 25030024, or equivalent), Penicillin-Streptomycin 100U/mL (GibcoTM, catalog number: 15140122, or equivalent).
- Trypsin-EDTA 0.05% phenol redGibco - Thermo FischerCatalog #25300054 (or equivalent)
- DPBS no calcium no magnesiumGibco - Thermo FischerCatalog #14190169
- Linear polyethylenimine (PEI MAX® - Transfection Grade Linear Polyethylenimine Hydrochloride (MW 40000)Polysciences, Inc.Catalog #24765-1 ); 1 mg/ml (w/v) stock in de-ionised H2O, pH 7.4; sterile filtered.
- Transfection media (for HEK293FT cells): Opti-MEM (Reduced Serum Medium)Thermo Fisher ScientificCatalog #31985062
- Plasmids for mammalian expression (pCMV vector):
FLAG-LRRK2 Y1699C (DU26486, available at MRCPPU Reagents and Services https://mrcppureagents.dundee.ac.uk)
RILPL1-GFP WT (DU27305, available at MRCPPU Reagents and Services https://mrcppureagents.dundee.ac.uk)
RILPL1-GFP R293A (DU68072, available at MRCPPU Reagents and Services https://mrcppureagents.dundee.ac.uk)
HA-Rab8A Q67L (DU51181, available at MRCPPU Reagents and Services https://mrcppureagents.dundee.ac.uk)
- Bradford Protein Assay Kit
- ChromoTek GFP-Trap® AgaroseProteintechCatalog # gta-20
- Lysis Buffer:
| A | |
| 50 mM Tris-HCl, pH 7.5 | |
| 10% (v/v) glycerol | |
| 150 mM NaCl | |
| 0.5 mM EDTA | |
| 1% (v/v) NP-40 Alternative (Merck #492016) | |
| 1X phosSTOP phosphatase inhibitor cocktail (PhosSTOP tablet: Roche, REF# 04906837001; to be added just before use) | |
| 1X protease inhibitor cocktail (cOmplete EDTA-free protease inhibitor cocktail tablet: Roche, REF# 11873580001; to be added just before use) |
- IP Wash Buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl
B. For TMT mass spectrometry analysis:
- DL-DithiothreitolMerck MilliporeSigma (Sigma-Aldrich)Catalog #D0632-10G – Prepare fresh as a 100 mM Stock in Milli-Q H2O
- IodoacetamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #I1149 – Prepare fresh as a 200 mM stock in Milli-Q H2O
- UreaThermo FisherCatalog #29700
- Elution buffer I (to be made just before use):
| A | B | |
| Urea | 2 M | |
| Tris-HCl pH 7.5 | 50 mM | |
| DTT | 1 mM |
- Elution buffer II (to be made just before use):
| A | B | |
| Urea | 2 M | |
| Tris-HCl pH 7.5 | 50 mM | |
| Iodoacetamide | 5 mM |
- TMT Isobaric Label Reagent Set (Thermo ScientificTM)
- LC-grade WaterFisher ScientificCatalog #10777404
- Triethylammonium bicarbonate bufferMerck MilliporeSigma (Sigma-Aldrich)Catalog #18597 – Make a 50 mM and 300 mM stock in LC-MS grade H2O, pH 8
- Seq Grade Modified Trypsin, 100ug (5 x 20ug)PromegaCatalog #V5111 – Make a stock by resuspending 20 µg trypsin in 0.05% (v/v) Acetic acid (just before use)
- LC-MS grade Methanol (MeOH) (Cat# 20847.307)
- LC-MS grade Acetonitrile (ACN) (Cat# 83640.320)
- Hydroxylamine solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #438227
- Trifluoroacetic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #T6508
- Formic acid (Sigma; Cat # 56302)
- Ammonium formateMerck MilliporeSigma (Sigma-Aldrich)Catalog #70221-25G-F
- Ammonium hydroxide solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #338818
- 2-propanol
- C18-HD bonded silica 12um particle size 47mm.CDS Analytical LLCCatalog #2215
- Empore Disk Strong Cation Exchange 12um particle size 47mmCDS Analytical LLCCatalog #Empore 2251
- pH indicator strips mid rangeVWR InternationalCatalog #1.09584.0001
- LC vials
Equipment
- CO2 Incubator for cell culture maintained at 37 °C, 5% CO2 (v/v).
- Laminar flow hood for cell culture.
- Refrigerated bench-top centrifuge (Eppendorf microcentrifuge 5417R, or equivalent).
- Plate reader for Protein quantification (BioTek Epoch, or equivalent)
- Thermo mixer (Eppendorf ThermoMixer, or equivalent)
Equipment
Savant™ SpeedVac™ Medium Capacity Vacuum Concentrators for Combinatorial Chemistry Applications
NAME
SpeedVac Vacuum Concentrator
TYPE
Thermo Scientific™
BRAND
SPD140P1
SKU
LINK
Consumables
- Falcon® 100 mm x 15 mm Not TC-treated Bacteriological Petri Dish 20/Pack 500/Case SterileCorningCatalog #351029
- SafeSeal reaction tube 1.5 ml PP PCR Performance Tested Low protein-bindingSarstedtCatalog #72.706.600
- Greiner Bio-One™ Polypropylene Pipette TipFisher ScientificCatalog #686271 and PIPETTE TIP 10 - 100 µL SUITABLE FOR EPPENDORF 96 PIECES / ST RACKgreiner bio-oneCatalog #685261 .
- Stripetter/stripette gun and stripettes
- Set of Gilson pipettes P10, P200, P1000
- 15 ml and 50 ml Falcons
- Stainless steel 316 syringe needle pipetting blunt 90° tipMerck MilliporeSigma (Sigma-Aldrich)Catalog #Z261378
- Glass pipettes (5 ml, 10 ml, 50 ml)
- C18 stage-tips (3 M Empore discs; C18 # 3M 2215 and SCX # 3M 2251)
DMEM high glucose no glutamineThermo Fisher ScientificCatalog #11960044
Fetal Bovine SerumMerck MilliporeSigma (Sigma-Aldrich)Catalog #F7524
L-Glutamine (200mM)Thermo Fisher ScientificCatalog #25030024
Penicillin-Streptomycin (10,000 U/mL)Gibco - Thermo FisherCatalog #15140122
NP-40 AlternativeMerck Millipore (EMD Millipore)Catalog #492016
Roche PhosSTOP™Merck MilliporeSigma (Sigma-Aldrich)Catalog #4906837001
cOmplete™ EDTA-free Protease Inhibitor CocktailRocheCatalog #11873580001
Transient transfection of HEK293 cells
Transient transfection of HEK293 cells
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 106 cells seeded per 10 cm dish).
Note
Note: For cells stably expressing the GFP-tagged protein of interest, proceed to Preparation and quantification of cell lysates (when cells are 90-100% confluent).
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 00:20:00 .
20m
Add the mixture dropwise to the cells from step 1 using a P1000 sterile pipette.
Incubate cells at 37 °C for 24:00:00 .
1d
Preparation and quantification of cell lysates
Preparation and quantification of cell lysates
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.
Note
Note: As HEK293 cells are loosely attached to the dish surface, extra care should be taken during the washing step.
Pour off media from the culture dish and completely aspirate any residual media.
Immediately add 400 µL 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 17000 x g, 4°C, 00:10:00 .
10m
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.
Note
Note: We recommend confirming the expression of the transiently expressed proteins by performing quantitative immunoblotting analysis as described in dx.doi.org/10.17504/protocols.io.bsgrnbv6.
Immunoprecipitation of GFP-tagged proteins from cell lysates
Immunoprecipitation of GFP-tagged proteins from cell lysates
Transfer n x 20 µL of packed ChromoTek GFP-Trap Agarose Beads (where n = number of samples) into a low binding Eppendorf tube.
Pellet the beads by centrifuging at 2500 x g, 4°C, 00:03:00 .
3m
Carefully aspirate the supernatant.
Resuspend the beads in 1 mL of IP wash buffer.
Repeat steps 18 to 20 twice.
Centrifuge at 2500 x g, 4°C, 00:03:00 and aspirate the supernatant.
3m
Resuspend beads from step 22 in n x 20 µL 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 (40 µL of slurry for each sample, corresponding to 20 µL of packed ChromoTek GFP-Trap Agarose Beads). Leave the tubes On ice until use.
For each sample, transfer 500 µg lysate from step 15 to the washed beads.
Incubate for 02:00:00 at 4 °C under mild agitation (on an orbital shaker).
2h
Pellet the beads by centrifuging at 2500 x g, 4°C, 00:05:00 .
5m
Carefully aspirate the supernatant.
Resuspend the beads in 1 mL of IP wash buffer.
Repeat steps 27 to 29 twice.
Centrifuge at 2500 x g, 4°C, 00:05:00 and aspirate the supernatant.
5m
Immediately proceed to Elution and on-bead tryptic digestion of immunoprecipitated proteins .
Elution and on-bead tryptic digestion of immunoprecipitated proteins
Elution and on-bead tryptic digestion of immunoprecipitated proteins
Add 100 µL of elution buffer I to the beads from step 32.
Add 500 ng of sequencing grade trypsin to the mixture and incubate on a Thermomixer at 800 rpm, 30°C, 00:30:00 .
Centrifuge the mixture at 2500 x g, Room temperature, 00:02:00 . Carefully transfer the supernatant to new Eppendorf tubes, being careful not to disturb the beads.
2m
Add 100 µL of elution buffer II to the beads from step 35 and mix gently by tapping. Centrifuge the mixture at 2500 x g, Room temperature, 00:02:00 . Carefully transfer the supernatant to the collection Eppendorf tubes from step 35, being careful not to disturb the beads.
2m
Incubate the Eppendorf tubes on the Thermomixer 800 rpm, 30°C Overnight (or minimum of 12 hr).
2h
Add 1% (v/v) Trifluoroacetic Acid (TFA) to the digested peptides from step 37. Incubate for 00:05:00 at Room temperature and centrifuge at 17000 x g, 00:10:00 .
15m
Peptide clean-up using C18 stage-tips
Peptide clean-up using C18 stage-tips
Prepare a C18 stage-tip for each sample as described in dx.doi.org/10.17504/protocols.io.bs3tngnn.
Note
A minimum of two discs are recommended for each 200 µL tip (assuming a peptide content of 5-10 µg).
C18 stage tips activation: add 80 µL of 100% ACN to each stage-tip and centrifuge at 2000 x g, 00:01:00 .
1m
C18 stage tips equilibration: add 80 µL of 0.1% TFA to each stage-tip and centrifuge at 2000 x g, 00:02:00 .
2m
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 2000 x g, 00:04:00 .
4m
Collect the flowthrough from step 44 and re-load onto the same C18 stage-tip. Centrifuge at 2000 x g, 00:04:00 .
4m
Wash the C18 stage-tips by adding 80 µL of 0.1% TFA and centrifuging at 2000 x g, 00:02:00 .
2m
Repeat step 46.
Transfer the C18 stage-tip to a new low-binding Eppendorf.
Add 30 µL of 30% (v/v) ACN in 0.1% (v/v) TFA to each stage-tip and centrifuge at 1000 x g, 00:01:00 .
1m
Repeat step 49.
Take 1-2 µL of the digested peptides, vacuum dry and inject on MS to verify the digestion efficiency.
Note
Note: Analyse data with a (1 h 10 mins) gradient run-on QE HF-X or Orbitrap Lumos mass spectrometer in a FT-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%.
Vacuum dry completely the remaining peptides and store at -80 °C until ready to undertake TMT labelling.
Tandem Mass Tag Labelling
Tandem Mass Tag Labelling
Dissolve 800 µg of each of the TMT mass tag reagents within the 11-plex TMT reagent kit in 40 µL of 100% anhydrous acetonitrile to obtain a 20 μg/μL concentration for each TMT reporter tag.
Leave at Room temperature for 00:10:00 , then vortex and spin 2000 x g, 00:02:00 .
Note
Note: Dissolved TMT reagents are prone to hydrolysis. Once reconstituted, aliquot and immediately transfer to -80 °C for storage (up to six months). Avoid multiple freeze thaw cycles.
12m
Dissolve lyophilized peptides from step 52 in 50 µL of a mixture containing 38 µL 50 millimolar (mM) TEAB buffer + 8 µL 100% (by vol) anhydrous acetonitrile.
Note
Note: It is important to maintain a final 30% (by vol) of anhydrous Acetonitrile for an effective TMT reaction.
Place the samples in a water bath sonicator for 00:10:00 .
10m
Centrifuge the samples 17000 x g, 00:10:00 .
10m
Transfer dissolved peptides into a 1.5ml protein low binding Eppendorf tube.
Add 10 µL 20 μg/μL TMT reagent i.e. 200 µg aiming for a 1:1 mass ratio of peptide: TMT reagent.
Give a gentle vortex and spin at 2000 x g, 00:01:00 .
1m
Place samples on a Thermomixer and incubate with gentle agitation at 800 rpm, Room temperature , 02:00:00 .
Add another 50 µL 50 millimolar (mM) TEAB buffer to make a final 100 µL reaction. Vortex, brief spin at 2000 x g, 00:10:00 and incubate on a Thermomixer for 00:10:00 .
Note
Note: It is good practice to maintain the total volume to 100 µL final reaction as this helps reducing pipetting error when aliquoting 5 µL of sample for label check efficiency.
20m
In order to verify the TMT labelling efficiency of each TMT mass tag, take a 5 µL 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 95 µL at -80 °C until the labelling efficiency has been verified.
Note
Note: It is important to verify the labelling efficiency of each TMT mass tag and it should label > 98%, by analysing on Mass spec. We recommend doing this employing a (2 h 25 min) FT-FT-MS2 study. This will establish that each reporter tag is efficiently labelled and ensure that an equal level of each peptide is labelled with each of the TMT tags. Search MS raw data with Proteome Discoverer 2.2 or 2.4 by enabling TMTreporter tag mass (+229.163 Da) on Lysine residue and Peptide N-terminus as dynamic modifications. Filter TMT labelled Peptide spectral matches (PSMs) in the modification tab to calculate the number of labelled and unlabelled PSMs to determine the labelling efficiency. Also, export PSM abundance in txt.file, to plot a Boxplot using R-software to determine the ~1:1 abundance within and between replicates.
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 5 µL 5% (by vol) Hydroxylamine to each sample to quench TMT reaction by incubating the reaction at Room temperature on a Thermomixer for 00:20:00 .
20m
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.
Note
Note: This can be used in case of sample loss during the downstream analysis or for further validation.
Snap freeze the remaining 80% of the reaction and vacuum dry using Speed vac.
Mini-basic RPLC fractionation
Mini-basic RPLC fractionation
Note
To improve the proteomic coverage of TMT labelled interactome, we recommend performing a stage-tip based mini-bRP fractionation (as described in [1]) by performing the following steps.
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 50 mL of bRP stock solution (50 millimolar (mM) Ammonium formate in Milli-Q H2O).
Prepare Solvent A: Mix 20 mL of bRP stock solution with 20 mL of Milli-Q H2O (=25 millimolar (mM) Ammonium formate in Milli-Q H2O).
Prepare Solvent B: Mix 20 mL of bRP stock solution with 20 mL of 100% Acetonitrile (=25 millimolar (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 200 µL of Solvent A.
Note
Note: Check the pH of the samples using a pH strip. Adjust to 10 by adding 0.5 µL of 30% Ammonium hydroxide solution if necessary.
Place samples on a Thermomixer at 1800 rpm, Room temperature , 00:20:00 .
Centrifuge sample at 17000 x g, Room temperature, 00:05:00 .
5m
Transfer the supernatant into a new 1.5ml protein low-binding Eppendorf tube.
Add 200 µL of 100% ACN to the C18 stage-tips (Step 71) and centrifuge at 2500 x g, Room temperature, 00:02:00 to activate the columns. Discard the flow through.
2m
Add 200 µL of Solvent B to each column from step 81 and centrifuge at 2500 x g, Room temperature, 00:02:00 . Discard the flow through.
2m
Add 200 µL of Solvent A to each column from step 82 and centrifuge at 2500 x g, Room temperature, 00:02:00 .
2m
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 1500 x g, Room temperature, 00:05:00 .
5m
Collect the flowthrough from step 86 and slowly load onto the same column.
Centrifuge at 1500 x g, Room temperature, 00:05:00 .
5m
Transfer the column into a new 1.5ml Eppendorf tube.
To wash the column, add 200 µL of Solvent A to the column and centrifuge at 2500 x g, Room temperature, 00:02:00 .
2m
Transfer the column into the tube labelled as “Fraction 1” (from step 72).
Add 60 µL of Elution solvent 1 (from step 76) to the column and centrifuge at 1500 x g, 00:02:00 .
2m
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 1500 x g, 00:02:00 .
2m
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.
LC-MS/MS analysis
LC-MS/MS analysis
Dissolve each fraction from step 95 in 20 µL of LC-buffer (3% (v/v) ACN, 0.1% (v/v) formic acid).
Place samples on a Thermomixer at 1800 rpm, Room temperature , 00:30:00 .
Transfer 10 µL 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 |
Protocol references
1. Ruprecht, B., et al., High pH Reversed-Phase Micro-Columns for Simple, Sensitive, and Efficient Fractionation of Proteome and (TMT labeled) Phosphoproteome Digests. Methods Mol Biol, 2017. 1550: p. 83-98.
2. dx.doi.org/10.17504/protocols.io.bs3tngnn
3. dx.doi.org/10.17504/protocols.io.bsgrnbv6