Jul 20, 2023
Sample preparation for TMT-based total and phospho-proteomic analysis of cells and tissues
- Ilham Seffouh1,
- Tran Le Cong Huyen Bao Phan1,
- Toan K. Phung1,
- Dario R Alessi1,
- Raja S. Nirujogi1
- 1Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK
Protocol Citation: Ilham Seffouh, Tran Le Cong Huyen Bao Phan, Toan K. Phung, Dario R Alessi, Raja S. Nirujogi 2023. Sample preparation for TMT-based total and phospho-proteomic analysis of cells and tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.261ged49yv47/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: July 14, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 85006
Keywords: Lysate preparation, Sample preparation for S-Trap assisted digestion, Sep-Pak purification, Phosphopeptide enrichment using TiO2, Tandem Mass Tags, Phosphoproteomic analysis, ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000463
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Abstract
Mass spectrometry-based proteomics and phosphoproteomics are highly sensitive and un-biased techniques to study the proteome and phosphoproteome at a global scale. Sample preparation is a key element for the generation of high quality, reproducible data. Here we provide a step-by-step protocol for processing material derived from cells or tissue samples. We recommend employing S-Trap assisted tryptic digestion followed by a TiO2-based phosphopeptide enrichment to achieve the highest possible reproducibility across experimental replicates. We also provide 10 or 16 plex Tandem Mass Tags (TMT) multiplexing strategy in combination with High-pH reversed-phase fractionation to achieve high coverage for phosphoproteomic analysis. The nano-liquid chromatography and High-resolution mass spectrometry instrument settings for both MS2 and Synchronous precursor selection MS3 data acquisition on Orbitrap Lumos Tribrid mass spectrometer are also described. Using these protocols, we routinely identify and quantify >35,000 phosphosites and ~10,000 protein groups.
Attachments
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Materials
Materials
Consumables:
- 1.5 ml protein low bind Eppendorf tubes (Eppendorf™ #022431081)
- 2 ml protein low bind Eppendorf tubes (Eppendorf™ #0030108132)
- Precellys Cryolys tissue homogenizer tubes (Precellys® Ceramic kit 2,8 mm, pre-filled with ceramic beads)
- 15 ml falcon tubes
- 15 ml racks
- Marker pen
- Pipette set (1 ml, 200 µl, 100 l, 20 l, 10 l)
- Pipette tips low binding (1 ml, 250 l, 10 l, Star labs Bevelled tips refill # S1111-3700, S1111-1706, S1111-6700)
- PPE kit (Lab coat, gloves, safety glasses)
- Dry ice
- Liquid Nitrogen
- Ice bucket
- 1.5 ml Eppendorf tubes rack
- 96 well plates - clear (Geneier Bio-one #655101)
- 2 ml tubes (Axygen™ MCT200C)
- 16-gauge needle (Sigma Aldrich # Z261378)
- X100 20 ml amber glass EPA vial with cap and seal (Thermo Scientific™ EPA Screw Vial Assembled Kit. Fisher Scientific # 11543750)**
- X72 40 ml amber glass EPA vial with cap and seal (Thermo Scientific™ EPA, TOC, and Scintillation Vials & Closures. Fisher Scientific # 12418656)**
- Millipore pH Strips (VWR # 1.09584.0001)
- CryoLys evolution homogenizer (Bertin technologies)
- Hard tissue homogenizing CK28 – 2 ml (CAT. NO.: P000911-LYSK0-A)
- S-Trap midi columns (https://www.protifi.com/)
- Sep-Pak Vac 1cc (50 mg) tC18-Cartridges (Waters # WAT054960)
- XBridge BEH C18 Column, 130A, 3.5 μ m, 4.6 x 250 mm (Waters # 186003943)
- 96 well 2.2 ml deep well plates (Fisher Scientific # 10089910)
- Evotips (EvoSep #EV2013 EVOTIP PURE, 10×96 TIPS)
- Acclaim PepMap 100 100 μm*cm nano viper trap column (Thermo Fisher Scientific # 164946)
- Easy-Spray PepMap RSLC C18 2 μm, 50 cm x 75 µm (Fisher Scientific #16692027)
Note
** Note: Prepare all stock and working reagents in these amber vials to store either at room temperature or at 4 °C depending on the reagent (store as per the protocol).
Reagents:
SDS Lysis Buffer: Final 2% (by mass) SDS in
| A | B | |
| Triethylammonium bicarbonate pH 8.5* | 100 mM | |
| sodium orthovanadate | 1 mM | |
| NaF | 50 mM | |
| b- glycerophosphate | 10 mM | |
| sodium pyrophosphate | 5 mM | |
| microcystin-LR | 1 μg/ml | |
| complete EDTA-free protease inhibitor cocktail (Roche) |
*TEABC, this is the natural pH of this buffer and made from a 1 M TEABC stock purchased from Sigma Catalogue number# T7408-500 ml.
- 20% (by mass) aqueous SDS stock
- BCA protein assay kit (Pierce # 23225)
- Tris (2-carboxyethyl) phosphine (TCEP) (Sigma Aldrich # 75259-10G).a
- Iodoacetamide (Sigma # I1149)
- LC-MS grade Trifluoroacetic acid (TFA) (Sigma# 302031-100 ML).b
- S-Trap protein binding buffer (90% (by vol) aqueous LC grade methanol containing a final concentration of 100 mM TEABC made from a 1 M TEABC stock purchased from Sigma Aldrich # T7408-500 ML)
- Sequencing grade trypsin (5 X 20 μg pack. Promega #V5111).c
- TPCK treated Trypsin from bovine pancreas (Sigma Aldrich # T1426-100MG)
- Methanol (VWR # 1.06035.2500)
- LC-MS grade Acetonitrile (VWR # 1.00030.2500)
- LC grade Formic acid (Sigma # 695076)
- Sep-Pak Purification: Activation buffer (100% Acetonitrile (ACN) (by vol)
- Sep-Pak Purification: Equilibration buffer (0.1% TFA (by vol) aqueous)
- Sep-Pak Purification: Wash buffer (0.1% formic acid (by vol) aqueous)
- Sep-Pak Purification: Elution buffer (0.1% formic acid (by vol) in 50% ACN (by vol) aqueous)
- Empore C18 disks, 47 mm (CDS analytical #2215)d
- High Select™ Phosphopeptide Enrichment Kits (Thermo Fisher Scientific #A32993)
- TMTpro™ 16plex Label Reagent Set (Thermo Fisher Scientific # A44520)
- Anhydrous Acetonitrile (Sigma Aldrich #271004)
- 50% (by vol) Hydroxylamine by mass (Sigma Aldrich # 467804)
- LC buffer (0.1% (by vol) Formic acid in 3% (by vol) Acetonitrile)
- Solvent-A1 (0.1% (by vol) TFA)
- Solvent-A2 (0.1% (by vol) Formic acid
- Solvent-B1 (50% (by vol) acetonitrile in 0.1% (by vol) TFA)
- Solvent-B2 (60% (by vol) acetonitrile in 0.1% (by vol) Formic acid)
Note
Notes:
a Prepare and store 10 µL aliquots of 1 M TCEP in Milli-Q H2O. Prior to use dilute the 1 Molarity (M) TCEP solution 10X in 300 millimolar (mM) TEABC to generate a stock solution of 0.1 Molarity (M) TCEP in 300 millimolar (mM) TEABC.
b Prepare 20% vol/vol stock in an amber bottle and store at 4 °C for up to six months. TFA is toxic, must be prepared in fume hood using a suitable glassware.
c Store stocks in -20 °C freezer and thaw trypsin stock just before the digestion step.
d Prepare a single layer with 16-gauge needle and pass it with spray duster into the 250 µL tip for 0.1 to 5 µg of peptide amount. For more than 5 µg use 2 or 3 layers of C18 material. Refer Figure 1 - see below for Stage-tip assembly.
Equipment:
- Pulveriser kit (https://cellcrusher.com/)
- -80 °C deep freezer, -20 °C freezer and 4 °C fridge
- Benchtop centrifuge (VWR)
- Eppendorf centrifuge
- Milli-Q water system
- Orbital shaker
- pH meter
- Plate reader for Protein quantification (BioTek Epoch)
- Diagenode Bioruptor plus sonication system
- Eppendorf Thermomixer with ThermoTop, 0.5 ml, 1.5 ml, 2 ml and 7 ml tubes compatible heating blocks
- Thermo Savant Speedvac system (#SPD140DDA)
- 1.5 ml tube floaters
- Branson water bath sonicator
- Dionex RSLC 3000 nano-LC system
- Dionex RSLC 3000 LC system for Offline fractionation with Auto sampler or Fraction collector, micro pump and VWD detector
- Orbitrap Fusion Lumos Tribrid Mass spectrometer
- Thermo Savant Speed vac system (#SPD140DDA)
- Nanodrop 1000 (Thermo Fisher Scientific)
- Rubber bulb # Fisher brand™ Rubber Pipette Bulb# 12446180
Protein LoBind tubesEppendorfCatalog #022431081
96-Well Microplate Flat Bottom non-sterile Polystyrene Clear 10/Pack 100/Casegreiner bio-oneCatalog #655101
Stainless steel 316 syringe needle pipetting blunt 90° tipMerck MilliporeSigma (Sigma-Aldrich)Catalog #Z261378
Thermo Scientific™ EPA Screw Vial Assembled Kit 20mL amber glass EPA vial with cap and sealThermo Fisher ScientificCatalog #11543750
Thermo Scientific™ EPA TOC and Scintillation Vials & ClosuresThermo Fisher ScientificCatalog #12418656
pH indicator strips mid rangeVWR InternationalCatalog #1.09584.0001
Hard Tissue homogenizing CK28BERTIN CORPCatalog #P000911-LYSK0-A
Sep-Pak tC18 1 cc Vac Cartridge 50 mg Sorbent per CartridgeWatersCatalog #WAT054960
XBridge BEH C18 Column 130Å 3.5 µm 4.6 mm X 250 mm 1/pkWatersCatalog #186003943
BRAND™ Deep Well PlatesFisher ScientificCatalog #10680763
EV2013 EVOTIP PURE 10×96 TIPSEVOSEPCatalog #EV2013
EASY-Spray™ C18 LC Columns, 2µm particle size, 250mm Length x 75µm I.D.Thermo FisherCatalog #ES802
Triethylammonium bicarbonate (TEAB)Merck MilliporeSigma (Sigma-Aldrich)Catalog #T7408
Pierce BCA Protein Assay Kit Thermo Fisher ScientificCatalog #23225
Tris(2-carboxyethyl)phosphine hydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #75259
IodoacetamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #I1149-5G
Trifluoroacetic acid for HPLC > 99.0%Merck MilliporeSigma (Sigma-Aldrich)Catalog #302031-100ML
Seq Grade Modified Trypsin, 100ug (5 x 20ug)PromegaCatalog #V5111
TPCK-trypsinMerck MilliporeSigma (Sigma-Aldrich)Catalog #T1426-50MG
Methanol LiChrosolv® hypergrade for LC-MS Supelco®VWR InternationalCatalog #1.06035.2500
Acetonitrile ≥99.9%VWR InternationalCatalog #1.00030.2500
LC-grade Formic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #695076
Empore™ Extraction Disc Anion C18CDCCatalog #2215
High-Select™ TiO2 Phosphopeptide Enrichment KitThermo FisherCatalog #A32993
Thermo Scientific™ TMTpro™ 16plex Label Reagent SetThermo Fisher ScientificCatalog #PIA44520
AcetonitrileMerck MilliporeSigma (Sigma-Aldrich)Catalog #271004
Hydroxylamine solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #467804
Lysate preparation: For cells
Lysate preparation: For cells
25m
25m
Prepare cells at a suitable confluency ~70 to 80% in a 15 cm dish. Ensure to have sufficient replicates, preferably 4 replicates per condition.
Note
Notes:
- The suitable starting material for an in-depth Phosphoproteomic analysis requires a minimum starting material of 3 mg protein amounts. If sufficient protein amounts not achievable from a single 15 cm dish, consider scaling up to pool from three 15 cm dishes per replicate in each condition.
- Phosphoproteomic sample preparation is lengthy and runs over a week period including several quality checks that need to be performed. It is possible that one or few samples may fail quality check, thus we recommend having a minimum of six replicates for each condition.
Wash cells with 5 mL plain DMEM medium and wash with 5 mL PBS.
Note
Note: All steps need to be performed with non-autoclaved low-binding pipette tips. This is to ensure not having any polymer contamination.
Add 700 µL of SDS lysis buffer to the dish and scrape it using a suitable scrapper, transfer the lysate into 1.5 ml low bind Eppendorf tube.
Boil samples at 95 °C for 00:05:00 , cool them On ice and subject samples to sonication using Bioruptor, 30 sec/ON and 30 sec/OFF per cycle for a total of 15 cycles.
Note
Note: If the protein lysate appears to be viscous, then consider using a probe sonicator.
5m
Centrifuge samples at 20000 x g, 00:20:00 and transfer the supernatant to a new 1.5 ml low bind Eppendorf tubes.
20m
Take an aliquot for protein estimation using BCA assay kit.
Note
Note: For cells we recommend having 1:10 dilution and to have standards with six points e.g., 125 μg/μL , 250 μg/μL , 500 μg/μL , 750 μg/μL , 1000 μg/μL , 1500 μg/μL , and 2000 μg/μL BSA as standards.
Transfer lysates to -80 °C freezer until further analysis.
Lysate preparation: For tissue samples
Lysate preparation: For tissue samples
2m
2m
Measure the wet weight of the tissue sample and always maintains samples on dry ice.
Transfer tissue samples to 2 mL Precellys Cryoyls-vials and add 1 mL of SDS lysis buffer.
Place vials in Precellys homogenizer and use a program with 3 cycles (2000 rpm for 30 sec ON and 20 sec Pause per cycle).
Centrifuge samples at 2000 x g, 00:02:00 .
Note
Note: Observe NO tissue chunks remain in the vial. If any, repeat homogenization for another 2 cycles.
2m
Transfer samples to new 1.5 ml low bind Eppendorf tubes and follow the steps described from step 4 to step 7.
Sample preparation for S-Trap assisted digestion
Sample preparation for S-Trap assisted digestion
18h 59m
18h 59m
Take 3 mg of protein for total and Phosphoproteomic analysis in a 2 ml low bind Eppendorf tubes.
Perform reduction by adding a 1 in 10 dilution of a solution of 0.1 Molarity (M) TCEP dissolved in 300 millimolar (mM) TEABC to bring final concentration of TCEP to 10 millimolar (mM) .
Incubate on a Thermomixer for 00:30:00 at 60 °C temperature with a gentle agitation.
30m
Bring tubes to Room temperature and add a 1 in 10 dilution of freshly prepared 0.4 Molarity (M) iodoacetamide dissolved in water.
Note
Note it is critical that the samples are at Room temperature prior to addition of iodoacetamide.
Incubate in dark on a Thermomixer at Room temperature for about 00:30:00 with a gentle agitation.
30m
Quench alkylation by addition of a 1 in 10 dilution of 0.1 Molarity (M) TCEP dissolved in 300 millimolar (mM) TEABC to bring final concentration of TCEP to 10 millimolar (mM) .
Incubate on a Thermomixer for 00:20:00 at Room temperature with a gentle agitation.
20m
Add SDS to a final concentration of 5% (by mass) from 20% (by mass) SDS stock.
Note
Note: 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).
Transfer lysates into a 15 ml falcon tube.
Add a final 1% (by vol) from a 20% (by vol) stock solution of Trifluoroacetic acid.
Dilute the samples to in 7 times the current volume of the mixture in of S-Trap wash buffer (90% (by vol) methanol in 0.1 Molarity (M) TEABC 7.1 v/v) (for examples if sample volume is 50 µL , add 300 µL of S-Trap wash buffer (90% (by vol) methanol in 0.1 Molarity (M) TEABC 7.1 (v/v)). Perform gentle vortex and transfer samples by pipetting up/down for few times to avoid any clumps.
Note
Note: We recommend processing a maximum of 24 samples at once. To avoid mistakes, number samples from 1 to 24 at every sub-sequent step.
Prepare an S-Trap midi column in a 15 ml falcon tube.
Add the diluted protein mixture to the column.
Centrifuge briefly to capture the protein particles at 2000 x g, Room temperature, 00:04:00 .
Note
Note: It is possible that the sample may not flowthrough completely. In such cases increase the centrifugation speed in a step-wise manner but not exceeding >4000 x g .
4m
Wash column with 3.5 mL of S-Trap buffer a total of 4 times (spin 2000 x g, 00:04:00 between washes).
Note
Note that the protein remains bound on the column and SDS and buffer components that affect trypsin digestion are removed.
4m
Move the S-Trap column to a clean 15 ml tube for digestion.
Add a 400 µL solution of freshly dissolved trypsin+Lys-C containing 30 µg for each sample freshly dissolved in 100 millimolar (mM) TEABC*. Simultaneously add 400 µL of TPCK treated trypsin in 100 millimolar (mM) TEABC containing 300 µg for each sample.
Centrifuge briefly at 200 x g, 00:01:00 .
1m
Collect flowthrough and reapply the trypsin solution back onto the column, being careful to avoid air bubbles.
Cap the tubes and incubate at 47 °C without shaking for 01:30:00 on a Thermomixer with a 15 ml heating block.
Note
Note: Do not shake as this causes bubbles and damage the column.
1h 30m
Incubate samples on Thermomixer for 16:00:00 at Room temperature .
Note
Note: Do not shake.
16h
Add 500 µL of 50 millimolar (mM) TEABC then spin to elute and place the eluate in a new 15ml falcon tube termed “eluate tube”.
Next, add 500 µL of 0.15% (by vol) Formic Acid and spin to elute. Also add this eluate to the “eluate tube”.
Finally, add 500 µL of 80% (by vol) Acetonitrile in 0.15% (by vol) formic acid and spin to elute. Also add this eluate to the “eluate tube”. Repeat this step two more times.
Note
Note 3 eluates should have been added to the eluate tube.
Take 1-2 µL of the combined eluate, vacuum dry and inject on MS to verify the digestion efficiency.
Note
Note: Analyse data with a 70 min 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 the remaining peptide amount and store in -80 °C deep freezer until the Sep-Pak purification.
Sep-Pak purification
Sep-Pak purification
44m
44m
Dissolve vacuum dried peptides by adding 1 mL of 1% TFA (by vol) aqueous and place the tubes on a Thermomixer at Room temperature for 00:30:00 shaking at 1800 rpm .
30m
Centrifuge tubes at high speed 17000 x g, Room temperature, 00:10:00 and place tubes aside for peptide purification using Sep-Pak cartridges.
10m
Place Sep-Pak Vac 1 cc (50 mg ) tC18 cartridges each in 15 ml falcon tubes.
Note
Note: The capacity of the Sep-Pak is ~5 to 8%, e.g. 50 mg cartridge can be used with up to 2-3 mg of peptide digest. One column wash equals to 1 cc = 1 ml of buffer.
Add 1 mL of Activation buffer (100% ACN by vol).
Centrifuge at 50 x g, Room temperature, 00:01:00 .
1m
Repeat step 42 for a total of 4 column washes and discard the flowthrough.
Add 1 mL of equilibration buffer (0.1% TFA (by vol) aqueous).
Centrifuge at 50 x g, Room temperature, 00:01:00 .
1m
Repeat step 45 for a total of 4 column washes and discard the flowthrough.
Load acidified peptide digest slowly onto the column.
Note
Note: DO NOT CENTRIFUGE. Let the column drain on gravity. If required, push the sample to drain one/two drops using rubber bulb.
Reapply the collected flowthrough onto the column and save the flowthrough.
Add 1 mL of wash buffer (0.1% formic acid (by vol) aqueous).
Centrifuge at 50 x g, Room temperature, 00:01:00 .
1m
Repeat step 50 for a total of 4 column washes and discard the flowthrough.
Place columns onto 1.5 ml low bind Eppendorf tubes for elution.
Note
Note: Use 200 µl pipette tip to place in between column and Eppendorf tube surface at the top such that the column can be lifted, not touching the bottom of the tube.
Add 350 µL of elution buffer (0.1% formic acid (by vol) in 50% ACN (by vol) aqueous). Let the buffer elute peptides by gravity.
Repeat step 54 for two more times. After final elution discard columns, vortex tubes and centrifuge at 17000 x g, Room temperature, 00:01:00 .
1m
Take 5% by vol for total proteomic analysis.
A small aliquot ~0.1% can be taken for the verification of tryptic digestion. Submit these samples for mass spectrometry (MS) analysis.
Snap freeze samples on dry-ice and vacuum dry using Speed Vac concentrator and store samples in -80 °C freezer until Phosphopeptide enrichment.
Phosphopeptide enrichment using TiO2
Phosphopeptide enrichment using TiO2
56m 30s
56m 30s
Label four sets of 2 ml low bind Eppendorf tubes.
Dissolve Sep-Pak purified peptide digest by adding 200 µL of binding buffer (provided with the kit). Place samples on a Thermomixer for 00:30:00 at Room temperature at 1800 rpm agitation.
30m
Centrifuge samples at 17000 x g, Room temperature, 00:05:00 and transfer supernatant to new 1.5 ml low bind Eppendorf tubes.
Note
Note: DO NOT collect any precipitate that may block TiO2 tips. Check peptide sample pH: pH should be < 3.0 .
5m
Take High-select Phosphopeptide enrichment kit (Thermo Fisher Scientific).
Note
Note: Equilibrate all solutions of the kit to room temperature prior to enrichment experiment (00:30:00 at Room temperature ). Securely tighten buffer bottle caps to prevent evaporation and store unused buffers and columns at 4 °C .
Label the TiO2 spin tips with a marker.
Note
Note: We recommend following 1 to 24 (if you are processing 24 samples). Place centrifuge column adaptor (provided with the kit) in a 2 ml low bind Eppendorf tubes and insert TiO2 spin tip into the adaptor.
Add 20 µL of Wash Buffer and centrifuge at 3000 x g, 00:02:00 .
Note
Note: All centrifugation steps for this protocol needs to be done at Room temperature .
2m
Add 20 µL of Binding/Equilibration Buffer and centrifuge at 3000 x g, 00:02:00 .
2m
Discard the flowthrough. Save the microcentrifuge tube for later "Wash column" step 1.
Transfer the equilibrated TiO2 spin tips along with the centrifuge column adaptor into a new 2 ml low bind Eppendorf tubes.
Apply 200 µL of suspended peptide sample to the spin tip. Centrifuge at 1000 x g, 00:05:00 .
5m
Reapply sample in the microcentrifuge tube to the spin tip. Centrifuge at 1000 x g, 00:05:00 .
Note
Note: If needed save the flowthrough for other PTM enrichment as Acetylation or Ubiquitinome analysis.
5m
Transfer the TiO2 spin tips along with the centrifuge column adaptor into a new 2 ml low bind Eppendorf tubes.
Wash column by adding 20 µL of Binding/Equilibration Buffer. Centrifuge at 3000 x g, 00:02:00 .
2m
Wash column by adding 20 µL of Wash Buffer. Centrifuge at 3000 x g, 00:02:00 .
2m
Repeat steps 71 and 72 in a sequential order.
Wash column by adding 20 µL of LC-MS grade water. Centrifuge at 3000 x g, 00:02:00 .
2m
Place TiO2 spin tips into new 2 ml low bind Eppendorf tubes. Add 60 µL of elution buffer and centrifuge at 1000 x g, 00:01:00 .
1m
Repeat step 75 for a second round of elution. Discard spin tips, vortex samples and centrifuge at 17000 x g, 00:00:30 .
30s
Take 1% of the sample for Phosphopeptide enrichment verification by MS analysis.
Take 25 % of the sample as a back-up or for Data Independent Acquisition (DIA)-based MS analysis.
Snap freeze samples on dry ice and subject them for vacuum dryness using Speed Vac concentrator.
The Phosphopeptides needs to be purified prior to the Tandem mass tags (TMT) labelling using Sep-Pak purification protocol described in section Sep-Pak purification. Follow all steps except use 200 µL of elution buffer and repeat elution two more times for a total of 600 µL of eluates.
Snap freeze samples on dry ice and subject them for vacuum dryness using Speed Vac concentrator. Store samples in -80 °C freezer until the TMT labelling.
Tandem Mass Tags (TMT) labelling of peptides
Tandem Mass Tags (TMT) labelling of peptides
3h 28m
3h 28m
Dissolve Sep-Pak purified total proteome and Phosphoproteomic samples by adding 30 µL of 50 millimolar (mM) TEABC buffer. Place samples on a Thermomixer at Room temperature with an agitation at 1800 rpm for 00:20:00 .
20m
Take out TMT kit from -80 °C freezer and equilibrate it to reach Room temperature .
Dissolve 800 µg of each of the TMT mass tag reagents within the 10 or 16-plex TMT reagent kit with 80 µL of 100% by vol anhydrous acetonitrile to obtain 10 μg/μL concentration for each TMT reporter tag.
Note
Note: Dissolved TMT reagents are prone to hydrolysis so immediately after aliquoting store remainder reagent in -80 °C deep freezer for long-term storage up to six months and try to avoid multiple freeze thaw cycles.
Transfer dissolved peptides into a 0.5 ml low bind Eppendorf tubes.
Add 20 µL of 10 μg/μL TMT reagent i.e., 200 µg .
Give a gentle vortex and brief spin 2000 x g, 00:01:00 .
1m
Place samples on a Thermomixer and incubate at Room temperature for 02:00:00 with a gentle agitation 800 rpm .
2h
Add another 50 µL of 50 millimolar (mM) TEABC buffer to make a final 100 µL reaction. Vortex, brief spin at 2000 x g, 00:01:00 and incubate on a Thermomixer for 00:10:00 .
Note
Note: It is a good practice to maintain the total volume to 100 µL final reaction as it helps in reducing pipetting error when aliquoting 5 µL of sample for label check efficiency.
11m
In order to verify the TMT labelling efficiency of each TMT mass tag, take a 5 µL aliquot from each of the TMT samples and pool this in a single tube and vacuum dry immediately using a Speed Vac.
Note
Note: It is important to verify the labelling efficiency of each TMT mass tag is and it should label > 98%, by analysing on Mass spec. We recommend doing this employing a 145 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 TMT-reporter 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. Alternatively, use in-house generated tool to normalise and adjust the volumes: https://samplepooler.proteo.info/).
Place remaining 95 µL of the reaction in -80 °C freezer. 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 91 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 of 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.
Take 20% of the reaction i.e. 220 µL (For 16 plex-TMT experiment take 320 µL ) as a backup, snap freeze on dry ice and vacuum dry.
Note
Note this is important because if there is a sample loss during the downstream analysis or to further validate.
Snap freeze the remaining 880 µL reaction and vacuum dry using Speed Vac.
Submit samples to MS facility for high pH fractionation.
Dissolve the digested peptide by adding 120 µL of High-pH Solvent-A (10 millimolar (mM) Ammonium formate 10.0 ). Place the sample on a Thermomixer with an agitation at 1800 rpm for 00:30:00 . Centrifuge at 17000 x g, 00:05:00 .
35m
Verify the pH to be ~ 10.0 . If pH appears to be low, adjust with Ammonium hydroxide (38% (by vol) by adding 1 µL and recheck the pH.
Ensure the LC-solvents are as Solvent-A (10 millimolar (mM) Ammonium formate 10.0 ); Solvent-B (90% ACN (v/v) in 10 millimolar (mM) Ammonium formate 10.0 ).
Note
Note: Adjust the pH with 30% Ammonium Hydroxide.
Prepare the LC method by following the below gradient:
| A | B | C | |
| Time (min) | Nano pump Flow rate (µl/min) | % of Solvent-B | |
| 0.0 | 0.275 | 3.0 | |
| 5.0 | 0.275 | 3.0 | |
| 20.0 | 0.100 | 3.0 | |
| 10.0 | 0.100 | 10.0 | |
| 50.0 | 0.100 | 40.0 | |
| 55.0 | 0.100 | 90.0 | |
| 62.0 | 0.100 | 90.0 | |
| 62.5 | 0.100 | 3.0 | |
| 70.0 | 0.100 | 3.0 | |
| 70.1 | 0.0100 | 3.0 |
Set the fraction collection time as Start time (min) 5.5 and End time (min) 62.0.
Collect a total of 96 fractions by keeping the fraction collection for 00:01:00 for each fraction.
1m
Concatenate by pooling distant fractions e.g. A1+D1, A2+D2, B1+E1, B2+E2 and so on to a total of 48 fractions in a 1.5 ml low bind Eppendorf tubes for LC-MS/MS analysis.
Snap freeze and vacuum dry using Speed Vac concentrator.
Prepare 2 µg of each fraction in 15 µL in LC buffer (0.1% (by vol) formic acid in 3% (by vol) Acetonitrile) and submit each fraction to the mass spectrometry facility.
Analyse each fraction by acquiring data in FT-FT-FT (MS3) HCD mode on a Orbitrap Fusion Lumos Mass spectrometer for 85 min run for each fraction.
LC-MS/MS analysis on Orbitrap Lumos Tribrid mass spectrometer for Phosphoproteomic analysis
LC-MS/MS analysis on Orbitrap Lumos Tribrid mass spectrometer for Phosphoproteomic analysis
Take 2 µg of each fraction from Phosphoproteomic experiment, transfer into LC vial and place it in LC autosampler tray.
Construct LC and MS method using the below settings.
LC Method: Dionex RSLC 3000 Ultimate LC system, 2 cm trap column and 50 cm analytical column connected and interfaced with Easy nano-source (Thermo Fisher Scientific).
| A | B | C | D | |
| No | Time (min) | Nano pump Flow rate (μl/min) | % Solvent-B | |
| 1 | 0 | 0.3 | 3 | |
| 2 | 5 | 0.3 | 8 | |
| 3 | 75 | 0.3 | 25 | |
| 4 | 85 | 0.3 | 35 | |
| 5 | 85.5 | 0.3 | 95 | |
| 6 | 93 | 0.3 | 95 | |
| 7 | 93.5 | 0.3 | 3 | |
| 8 | 100 | 0.3 | 3 | |
| 9 | 100 | Stop |
Mass spectrometer parameters: Refer below settings to construct FT-FT-HCD (MS2) method:
| A | B | |
| Method Summary | ||
| Method Settings | ||
| Application Mode | Peptide | |
| Method Duration (min) | 100 | |
| Global Parameters | ||
| Ion Source | ||
| Use Ion Source Settings from Tune | True | |
| FAIMS Mode | Not Installed | |
| MS Global Settings | ||
| Infusion Mode | Liquid Chromatography | |
| Expected LC Peak Width (s) | 30 | |
| Advanced Peak Determination | True | |
| Default Charge State | 2 | |
| Internal Mass Calibration | Off | |
| Experiment#1 [MS] | ||
| Start Time (min) | 0 | |
| End Time (min) | 100 | |
| Master Scan | ||
| MS OT | ||
| Detector Type | Orbitrap | |
| Orbitrap Resolution | 120000 | |
| Mass Range | Normal | |
| Use Quadrupole Isolation | True | |
| Scan Range (m/z) | 375-1400 | |
| RF Lens (%) | 32 | |
| AGC Target | Standard | |
| 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+04 | |
| Precursor Fit | ||
| Fit Threshold (%) | 70 | |
| Fit Window (m/z) | 0.7 | |
| Data Dependent | ||
| Data Dependent Mode | Number of Scans | |
| Number of Dependent Scans | 15 | |
| 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 (%) | 30 | |
| Detector Type | Orbitrap | |
| Orbitrap Resolution | 50000 | |
| Mass Range | Normal | |
| Scan Range Mode | Define First Mass | |
| First Mass (m/z) | 110 | |
| 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 |
Export the MS raw data for database searches using MaxQuant or MS-Fragger. Analyse database search results using Perseus software package or R or MS-Stats or Python for statistical analysis.
LC-MS/MS analysis on Orbitrap Lumos Tribrid mass spectrometer for total proteomic analysis
LC-MS/MS analysis on Orbitrap Lumos Tribrid mass spectrometer for total proteomic analysis
Take 2 µg of each fraction from Phosphoproteomics experiment, transfer into LC vial and place it in LC autosampler tray.
Construct LC and MS method using the below settings.
LC Method: Dionex RSLC 3000 Ultimate LC system, 2 cm trap column and 50 cm analytical column connected and interfaced with Easy nano-source (Thermo Fisher Scientific).
| A | B | C | D | |
| No | Time (min) | Nano pump Flow rate (μl/min) | % Solvent-B | |
| 1 | 0 | 0.3 | 3 | |
| 2 | 5 | 0.3 | 8 | |
| 3 | 7 | 0.3 | 25 | |
| 4 | 85 | 0.3 | 35 | |
| 5 | 86 | 0.3 | 95 | |
| 6 | 92 | 0.3 | 95 | |
| 7 | 93 | 0.3 | 3 | |
| 8 | 100 | 0.3 | 3 | |
| 9 | 100 | Stop | ||
Mass spectrometer parameters: Refer below settings to construct FT-IT-HCD-FT-HCD (MS3) method:
| A | B | |
| Method Summary | ||
| Method Settings | ||
| Application Mode | Peptide | |
| Method Duration (min) | 100 | |
| Global Parameters | ||
| Ion Source | ||
| Use Ion Source Settings from Tune | True | |
| FAIMS Mode | Not Installed | |
| MS Global Settings | ||
| Infusion Mode | Liquid Chromatography | |
| Expected LC Peak Width (s) | 30 | |
| Advanced Peak Determination | True | |
| Default Charge State | 2 | |
| Internal Mass Calibration | Off | |
| Experiment#1 [MS] | ||
| Start Time (min) | 0 | |
| End Time (min) | 100 | |
| Cycle Time (sec) | 2 | |
| 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 | Standard | |
| 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 | |
| Precursor Fit | ||
| Fit Threshold (%) | 70 | |
| Fit Window (m/z) | 0.7 | |
| Data Dependent | ||
| Data Dependent Mode | Cycle Time | |
| Time between Master Scans (sec) | 2 | |
| Scan Event Type 1 | ||
| Scan | ||
| ddMS² IT HCD | ||
| Isolation Mode | Quadrupole | |
| Isolation Window (m/z) | 0.7 | |
| Isolation Offset | Off | |
| Activation Type | HCD | |
| Collision Energy Mode | Fixed | |
| HCD Collision Energy (%) | 32 | |
| Detector Type | Ion Trap | |
| Ion Trap Scan Rate | Rapid | |
| Mass Range | Normal | |
| Scan Range Mode | Define m/z range | |
| Scan Range (m/z) | 200-1400 | |
| AGC Target | Custom | |
| Normalized AGC Target (%) | 200 | |
| Maximum Injection Time Mode | Custom | |
| Maximum Injection Time (ms) | 50 | |
| Micro scans | 1 | |
| Data Type | Centroid | |
| Scan Description | ||
| Filters | ||
| Precursor Selection Range | ||
| Selection Range Mode | Mass Range | |
| Mass Range (m/z) | 400-1400 | |
| Precursor Ion Exclusion | ||
| Exclusion mass width | ppm | |
| Low | 25 | |
| High | 25 | |
| Isobaric Tag Loss Exclusion | ||
| Reagent | TMTpro | |
| 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 | 10 | |
| MS Isolation Window (m/z) | 0.7 | |
| MS2 Isolation Window (m/z) | 2 | |
| Isolation Offset | Off | |
| Activation Type | HCD | |
| HCD Collision Energy (%) | 55 | |
| Detector Type | Orbitrap | |
| Orbitrap Resolution | 50000 | |
| Mass Range | Normal | |
| Scan Range Mode | Define m/z range | |
| Scan Range (m/z) | 110-500 | |
| AGC Target | Standard | |
| 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 | 10 |
Export the MS raw data for database searches using MaxQuant or MS-Fragger. Analyse database search results using Perseus software package or R or MS-Stats or Python for statistical analysis.