Sample preparation for TMT-based total and phospho-proteomic analysis of cells and tissues

Dario R Alessi, Toan K. Phung, Raja S. Nirujogi, Ilham Seffouh, Tran Le Cong Huyen Bao Phan

Published: 2023-07-21 DOI: 10.17504/protocols.io.261ged49yv47/v1

Sample preparation for S-Trap assisted digestion

Phosphopeptide enrichment using TiO2

Disclaimer

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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

787-2015.docx

Steps

Lysate preparation: For cells

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 5mL plain DMEM medium and wash with 5mL 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 700µL 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 0h 5m 0s, 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.

Centrifuge samples at 20000x g and transfer the supernatant to a new 1.5 ml low bind Eppendorf tubes.

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., 125μg/μL, 250μg/μL, 500μg/μL, 750μg/μL, 1000μg/μL, 1500μg/μL, and 2000μg/μL BSA as standards.

Transfer lysates to -80°C freezer until further analysis.

Lysate preparation: For tissue samples

Measure the wet weight of the tissue sample and always maintains samples on dry ice.

Transfer tissue samples to 2mL Precellys Cryoyls-vials and add 1mL of SDS lysis buffer.

10.

Place vials in Precellys homogenizer and use a program with 3 cycles (2000rpm for 30 sec ON and 20 sec Pause per cycle).

11.

Centrifuge samples at 2000x g.

Note

Note : Observe NO tissue chunks remain in the vial. If any, repeat homogenization for another 2 cycles.

12.

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

13.

Take 3mg of protein for total and Phosphoproteomic analysis in a 2 ml low bind Eppendorf tubes.

14.

Perform reduction by adding a 1 in 10 dilution of a solution of 0.1Molarity (M) TCEP dissolved in 300millimolar (mM) TEABC to bring final concentration of TCEP to 10millimolar (mM).

15.

Incubate on a Thermomixer for 0h 30m 0s at 60°C temperature with a gentle agitation.

16.

Bring tubes to Room temperature and add a 1 in 10 dilution of freshly prepared 0.4Molarity (M) iodoacetamide dissolved in water.

Note

Note it is critical that the samples are at Room temperature prior to addition of iodoacetamide.

17.

Incubate in dark on a Thermomixer at Room temperature for about 0h 30m 0s with a gentle agitation.

18.

Quench alkylation by addition of a 1 in 10 dilution of 0.1Molarity (M) TCEP dissolved in 300millimolar (mM) TEABC to bring final concentration of TCEP to 10millimolar (mM).

19.

Incubate on a Thermomixer for 0h 20m 0s at Room temperature with a gentle agitation.

20.

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).

21.

Transfer lysates into a 15 ml falcon tube.

22.

Add a final 1% (by vol) from a 20% (by vol) stock solution of Trifluoroacetic acid.

23.

Dilute the samples to in 7 times the current volume of the mixture in of S-Trap wash buffer (90% (by vol) methanol in 0.1Molarity (M) TEABC 7.1 v/v) (for examples if sample volume is 50µL, add 300µL of S-Trap wash buffer (90% (by vol) methanol in 0.1Molarity (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.

24.

Prepare an S-Trap midi column in a 15 ml falcon tube.

25.

Add the diluted protein mixture to the column.

26.

Centrifuge briefly to capture the protein particles at 2000x g.

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 >4000x g,undefine.

27.

Wash column with 3.5mL of S-Trap buffer a total of 4 times (spin 2000x g between washes).

Note

Note that the protein remains bound on the column and SDS and buffer components that affect trypsin digestion are removed.

28.

Move the S-Trap column to a clean 15 ml tube for digestion.

29.

Add a 400µL solution of freshly dissolved trypsin+Lys-C containing 30µg for each sample freshly dissolved in 100millimolar (mM) TEABC*. Simultaneously add 400µL of TPCK treated trypsin in 100millimolar (mM) TEABC containing 300µg for each sample.

30.

Centrifuge briefly at 200x g.

31.

Collect flowthrough and reapply the trypsin solution back onto the column, being careful to avoid air bubbles.

32.

Cap the tubes and incubate at 47°C without shaking for 1h 30m 0s on a Thermomixer with a 15 ml heating block.

Note

Note: Do not shake as this causes bubbles and damage the column.

33.

Incubate samples on Thermomixer for 16h 0m 0s at Room temperature.

Note

Note: Do not shake.

34.

Add 500µL of 50millimolar (mM) TEABC then spin to elute and place the eluate in a new 15ml falcon tube termed “eluate tube”.

35.

Next, add 500µL of 0.15% (by vol) Formic Acid and spin to elute. Also add this eluate to the “eluate tube”.

36.

Finally, add 500µL 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.

37.

Take 1-2µL 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%.

38.

Vacuum dry the remaining peptide amount and store in -80°C deep freezer until the Sep-Pak purification.

Sep-Pak purification

39.

Dissolve vacuum dried peptides by adding 1mL of 1% TFA (by vol) aqueous and place the tubes on a Thermomixer at Room temperature for 0h 30m 0s shaking at 1800rpm.

40.

Centrifuge tubes at high speed 17000x g and place tubes aside for peptide purification using Sep-Pak cartridges.

41.

Place Sep-Pak Vac 1 cc (50mg) tC18 cartridges each in 15 ml falcon tubes.

Note

Note : The capacity of the Sep-Pak is ~5 to 8%, e.g. 50mg cartridge can be used with up to 2-3mg of peptide digest. One column wash equals to 1 cc = 1 ml of buffer.

42.

Add 1mL of Activation buffer (100% ACN by vol).

43.

Centrifuge at 50x g.

44.

Repeat step 42 for a total of 4 column washes and discard the flowthrough.

45.

Add 1mL of equilibration buffer (0.1% TFA (by vol) aqueous).

46.

Centrifuge at 50x g.

47.

Repeat step 45 for a total of 4 column washes and discard the flowthrough.

48.

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.

49.

Reapply the collected flowthrough onto the column and save the flowthrough.

50.

Add 1mL of wash buffer (0.1% formic acid (by vol) aqueous).

51.

Centrifuge at 50x g.

52.

Repeat step 50 for a total of 4 column washes and discard the flowthrough.

53.

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.

54.

Add 350µL of elution buffer (0.1% formic acid (by vol) in 50% ACN (by vol) aqueous). Let the buffer elute peptides by gravity.

55.

Repeat step 54 for two more times. After final elution discard columns, vortex tubes and centrifuge at 17000x g.

56.

Take 5% by vol for total proteomic analysis.

57.

A small aliquot ~0.1% can be taken for the verification of tryptic digestion. Submit these samples for mass spectrometry (MS) analysis.

58.

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

59.

Label four sets of 2 ml low bind Eppendorf tubes.

60.

Dissolve Sep-Pak purified peptide digest by adding 200µL of binding buffer (provided with the kit). Place samples on a Thermomixer for 0h 30m 0s at Room temperature at 1800rpm agitation.

61.

Centrifuge samples at 17000x g 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.

62.

Take High-select Phosphopeptide enrichment kit (Thermo Fisher Scientific).

Note

Note : Equilibrate all solutions of the kit to room temperature prior to enrichment experiment (0h 30m 0s at Room temperature). Securely tighten buffer bottle caps to prevent evaporation and store unused buffers and columns at 4°C.

63.

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.

64.

Add 20µL of Wash Buffer and centrifuge at 3000x g.

Note

Note : All centrifugation steps for this protocol needs to be done at Room temperature.

65.

Add 20µL of Binding/Equilibration Buffer and centrifuge at 3000x g.

66.

Discard the flowthrough. Save the microcentrifuge tube for later "Wash column" step 1.

67.

Transfer the equilibrated TiO₂ spin tips along with the centrifuge column adaptor into a new 2 ml low bind Eppendorf tubes.

68.

Apply 200µL of suspended peptide sample to the spin tip. Centrifuge at 1000x g.

69.

Reapply sample in the microcentrifuge tube to the spin tip. Centrifuge at 1000x g.

Note

Note : If needed save the flowthrough for other PTM enrichment as Acetylation or Ubiquitinome analysis.

70.

Transfer the TiO₂ spin tips along with the centrifuge column adaptor into a new 2 ml low bind Eppendorf tubes.

71.

Wash column by adding 20µL of Binding/Equilibration Buffer. Centrifuge at 3000x g.

72.

Wash column by adding 20µL of Wash Buffer. Centrifuge at 3000x g.

73.

Repeat steps 71 and 72 in a sequential order.

74.

Wash column by adding 20µL of LC-MS grade water. Centrifuge at 3000x g.

75.

Place TiO₂ spin tips into new 2 ml low bind Eppendorf tubes. Add 60µL of elution buffer and centrifuge at 1000x g.

76.

Repeat step 75 for a second round of elution. Discard spin tips, vortex samples and centrifuge at 17000x g.

77.

Take 1% of the sample for Phosphopeptide enrichment verification by MS analysis.

78.

Take 25 % of the sample as a back-up or for Data Independent Acquisition (DIA)-based MS analysis.

79.

Snap freeze samples on dry ice and subject them for vacuum dryness using Speed Vac concentrator.

80.

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 200µL of elution buffer and repeat elution two more times for a total of 600µL of eluates.

81.

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

82.

Dissolve Sep-Pak purified total proteome and Phosphoproteomic samples by adding 30µL of 50millimolar (mM) TEABC buffer. Place samples on a Thermomixer at Room temperature with an agitation at 1800rpm for 0h 20m 0s.

83.

Take out TMT kit from -80°C freezer and equilibrate it to reach Room temperature.

84.

Dissolve 800µg of each of the TMT mass tag reagents within the 10 or 16-plex TMT reagent kit with 80µL of 100% by vol anhydrous acetonitrile to obtain 10μg/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.

85.

Transfer dissolved peptides into a 0.5 ml low bind Eppendorf tubes.

86.

Add 20µL of 10μg/μL TMT reagent i.e., 200µg.

87.

Give a gentle vortex and brief spin 2000x g.

88.

Place samples on a Thermomixer and incubate at Room temperature for 2h 0m 0s with a gentle agitation 800rpm.

89.

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

Note

Note : It is a good practice to maintain the total volume to 100µL final reaction as it helps in reducing pipetting error when aliquoting 5µL of sample for label check efficiency.

90.

In order to verify the TMT labelling efficiency of each TMT mass tag, take a 5µL 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/).

91.

Place remaining 95µL 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.

92.

Thaw stored TMT labelled samples from step 91 to Room temperature.

93.

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

94.

Add 5µL of 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.

95.

Pool all samples into a single tube.

96.

Take 20% of the reaction i.e. 220µL (For 16 plex-TMT experiment take 320µL) 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.

97.

Snap freeze the remaining 880µL reaction and vacuum dry using Speed Vac.

98.

Submit samples to MS facility for high pH fractionation.

99.

Dissolve the digested peptide by adding 120µL of High-pH Solvent-A (10millimolar (mM) Ammonium formate 10.0). Place the sample on a Thermomixer with an agitation at 1800rpm for 0h 30m 0s. Centrifuge at 17000x g.

100.

Verify the pH to be ~ 10.0. If pH appears to be low, adjust with Ammonium hydroxide (38% (by vol) by adding 1µL and recheck the pH.

101.

Ensure the LC-solvents are as Solvent-A (10millimolar (mM) Ammonium formate 10.0); Solvent-B (90% ACN (v/v) in 10millimolar (mM) Ammonium formate 10.0).

Note

Note : Adjust the pH with 30% Ammonium Hydroxide.

102.

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

103.

Set the fraction collection time as Start time (min) 5.5 and End time (min) 62.0.

104.

Collect a total of 96 fractions by keeping the fraction collection for 0h 1m 0s for each fraction.

105.

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.

106.

Snap freeze and vacuum dry using Speed Vac concentrator.

107.

Prepare 2µg of each fraction in 15µL in LC buffer (0.1% (by vol) formic acid in 3% (by vol) Acetonitrile) and submit each fraction to the mass spectrometry facility.

108.

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

109.

Take 2µg of each fraction from Phosphoproteomic experiment, transfer into LC vial and place it in LC autosampler tray.

110.

Construct LC and MS method using the below settings.

111.

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

112.

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

113.

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

114.

Take 2µg of each fraction from Phosphoproteomics experiment, transfer into LC vial and place it in LC autosampler tray.

115.

Construct LC and MS method using the below settings.

116.

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

117.

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

118.

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.

Sample preparation for TMT-based total and phospho-proteomic analysis of cells and tissues

Disclaimer

Abstract

Attachments

Steps

Lysate preparation: For cells

Lysate preparation: For tissue samples

Sample preparation for S-Trap assisted digestion

Sep-Pak purification

Phosphopeptide enrichment using TiO2

Tandem Mass Tags (TMT) labelling of peptides

LC-MS/MS analysis on Orbitrap Lumos Tribrid mass spectrometer for Phosphoproteomic analysis

LC-MS/MS analysis on Orbitrap Lumos Tribrid mass spectrometer for total proteomic analysis

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