TITLE: Sample preparation for TMT-based total and phospho-proteomic analysis of cells and tissues
AUTHORS: Ilham Seffouh, Tran Le Cong Huyen Bao Phan, Toan K. Phung, Dario R Alessi, Raja S. Nirujogi

[DESCRIPTION]
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.

[STEPS]
SECTION: Lysate preparation: For cells
1. Prepare cells at a suitable confluency ~70 to 80% in a 15 cm dish. Ensure to have sufficient replicates, preferably 4 replicates per condition. 
 

SECTION: Lysate preparation: For cells
2. Wash cells with 5 mL plain DMEM medium and wash with 5 mL  PBS. 
 

SECTION: Lysate preparation: For cells
3. 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.
SECTION: Lysate preparation: For cells
4. Boil samples at 95 °C for 5 min, 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. 
 

SECTION: Lysate preparation: For cells
5. Centrifuge samples at 20000 x g, 20 min and transfer the supernatant to a new 1.5 ml low bind Eppendorf tubes.
SECTION: Lysate preparation: For cells
6. Take an aliquot for protein estimation using BCA assay kit. 
 

SECTION: Lysate preparation: For cells
7. Transfer lysates to -80 °C freezer until further analysis.
SECTION: Lysate preparation: For tissue samples
8. Measure the wet weight of the tissue sample and always maintains samples on dry ice.

SECTION: Lysate preparation: For tissue samples
9. Transfer tissue samples to 2 mL Precellys Cryoyls-vials and add 1 mL of SDS lysis buffer.

SECTION: Lysate preparation: For tissue samples
10. Place vials in Precellys homogenizer and use a program with 3 cycles (  for 30 sec ON and 20 sec Pause per cycle).
SECTION: Lysate preparation: For tissue samples
11. Centrifuge samples at 2000 x g, 2 min. 
 


SECTION: Lysate preparation: For tissue samples
12. Transfer samples to new 1.5 ml low bind Eppendorf tubes and follow the steps described from step 4 to step 7.
SECTION: Sample preparation for S-Trap assisted digestion
13. Take 3 mg of protein for total and Phosphoproteomic analysis in a 2 ml low bind Eppendorf tubes.
SECTION: Sample preparation for S-Trap assisted digestion
14. 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).

SECTION: Sample preparation for S-Trap assisted digestion
15. Incubate on a Thermomixer for 30 min at 60 °C temperature with a gentle agitation.

SECTION: Sample preparation for S-Trap assisted digestion
16. Bring tubes to Room temperature and add a 1 in 10 dilution of freshly prepared 0.4 Molarity (M) iodoacetamide dissolved in water. 
 

SECTION: Sample preparation for S-Trap assisted digestion
17. Incubate in dark on a Thermomixer at Room temperature for about 30 min with a gentle agitation.
SECTION: Sample preparation for S-Trap assisted digestion
18. 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).
SECTION: Sample preparation for S-Trap assisted digestion
19. Incubate on a Thermomixer for 20 min at Room temperature with a gentle agitation.
SECTION: Sample preparation for S-Trap assisted digestion
20. Add SDS to a final concentration of 5% (by mass) from 20% (by mass) SDS stock.  
 

SECTION: Sample preparation for S-Trap assisted digestion
21. Transfer lysates into a 15 ml falcon tube.
SECTION: Sample preparation for S-Trap assisted digestion
22. Add a final 1% (by vol) from a 20% (by vol) stock solution of Trifluoroacetic acid.
SECTION: Sample preparation for S-Trap assisted digestion
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 0.1 Molarity (M) TEABC pH 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 pH 7.1 (v/v)). Perform gentle vortex and transfer samples by pipetting up/down for few times to avoid any clumps. 
 

SECTION: Sample preparation for S-Trap assisted digestion
24. Prepare an S-Trap midi column in a 15 ml falcon tube.
SECTION: Sample preparation for S-Trap assisted digestion
25. Add the diluted protein mixture to the column.
SECTION: Sample preparation for S-Trap assisted digestion
26. Centrifuge briefly to capture the protein particles at 2000 x g, 4 min. 
 

SECTION: Sample preparation for S-Trap assisted digestion
27. Wash column with 3.5 mL of S-Trap buffer a total of 4 times (spin 2000 x g, 4 min between washes). 
 

SECTION: Sample preparation for S-Trap assisted digestion
28. Move the S-Trap column to a clean 15 ml tube for digestion.
SECTION: Sample preparation for S-Trap assisted digestion
29. 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.


SECTION: Sample preparation for S-Trap assisted digestion
30. Centrifuge briefly at 200 x g, 1 min.

SECTION: Sample preparation for S-Trap assisted digestion
31. Collect flowthrough and reapply the trypsin solution back onto the column, being careful to avoid air bubbles.
SECTION: Sample preparation for S-Trap assisted digestion
32. Cap the tubes and incubate at 47 °C without shaking for 90 min on a Thermomixer with a 15 ml heating block. 
 

SECTION: Sample preparation for S-Trap assisted digestion
33. Incubate samples on Thermomixer for 960 min at Room temperature. 
 


SECTION: Sample preparation for S-Trap assisted digestion
34. 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”.
SECTION: Sample preparation for S-Trap assisted digestion
35. Next, add 500 µL of 0.15% (by vol) Formic Acid and spin to elute. Also add this eluate to the “eluate tube”.
SECTION: Sample preparation for S-Trap assisted digestion
36. 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.
 

SECTION: Sample preparation for S-Trap assisted digestion
37. Take 1-2 µL of the combined eluate, vacuum dry and inject on MS to verify the digestion efficiency. 
 

SECTION: Sample preparation for S-Trap assisted digestion
38. Vacuum dry the remaining peptide amount and store in -80 °C deep freezer until the Sep-Pak purification.
SECTION: Sep-Pak purification
39. 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 30 min shaking at  .
SECTION: Sep-Pak purification
40. Centrifuge tubes at high speed 17000 x g, 10 min and place tubes aside for peptide purification using Sep-Pak cartridges.
SECTION: Sep-Pak purification
41. Place Sep-Pak Vac 1 cc (50 mg) tC18 cartridges each in 15 ml falcon tubes. 
 

SECTION: Sep-Pak purification
42. Add 1 mL of Activation buffer (100% ACN by vol).
SECTION: Sep-Pak purification
43. Centrifuge at 50 x g, 1 min.

SECTION: Sep-Pak purification
44. Repeat step 42 for a total of 4 column washes and discard the flowthrough.
SECTION: Sep-Pak purification
45. Add 1 mL of equilibration buffer (0.1% TFA (by vol) aqueous).
SECTION: Sep-Pak purification
46. Centrifuge at 50 x g, 1 min.
SECTION: Sep-Pak purification
47. Repeat step 45 for a total of 4 column washes and discard the flowthrough.
SECTION: Sep-Pak purification
48. Load acidified peptide digest slowly onto the column. 
 

SECTION: Sep-Pak purification
49. Reapply the collected flowthrough onto the column and save the flowthrough.
SECTION: Sep-Pak purification
50. Add 1 mL of wash buffer (0.1% formic acid (by vol) aqueous).

SECTION: Sep-Pak purification
51. Centrifuge at  50 x g, 1 min.
SECTION: Sep-Pak purification
52. Repeat step 50 for a total of 4 column washes and discard the flowthrough.
SECTION: Sep-Pak purification
53. Place columns onto 1.5 ml low bind Eppendorf tubes for elution. 
 

SECTION: Sep-Pak purification
54. 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.
SECTION: Sep-Pak purification
55. Repeat step 54 for two more times. After final elution discard columns, vortex tubes and centrifuge at 17000 x g, 1 min.

SECTION: Sep-Pak purification
56. Take 5% by vol for total proteomic analysis.
SECTION: Sep-Pak purification
57. A small aliquot ~0.1% can be taken for the verification of tryptic digestion. Submit these samples for mass spectrometry (MS) analysis.
SECTION: Sep-Pak purification
58. Snap freeze samples on dry-ice and vacuum dry using Speed Vac concentrator and store samples in -80 °C freezer until Phosphopeptide enrichment.
SECTION: Phosphopeptide enrichment using TiO2
59. Label four sets of 2 ml low bind Eppendorf tubes.

SECTION: Phosphopeptide enrichment using TiO2
60. Dissolve Sep-Pak purified peptide digest by adding 200 µL of binding buffer (provided with the kit). Place samples on a Thermomixer for 30 min at Room temperature at   agitation.
SECTION: Phosphopeptide enrichment using TiO2
61. Centrifuge samples at 17000 x g, 5 min and transfer supernatant to new 1.5 ml low bind Eppendorf tubes.
 

SECTION: Phosphopeptide enrichment using TiO2
62. Take High-select Phosphopeptide enrichment kit (Thermo Fisher Scientific).
 

SECTION: Phosphopeptide enrichment using TiO2
63. Label the TiO2 spin tips with a marker.
 
 
SECTION: Phosphopeptide enrichment using TiO2
64. Add 20 µL of Wash Buffer and centrifuge at 3000 x g, 2 min. 
 

SECTION: Phosphopeptide enrichment using TiO2
65. Add 20 µL of Binding/Equilibration Buffer and centrifuge at 3000 x g, 2 min.

SECTION: Phosphopeptide enrichment using TiO2
66. Discard the flowthrough. Save the microcentrifuge tube for later "Wash column" step 1.
SECTION: Phosphopeptide enrichment using TiO2
67. Transfer the equilibrated TiO2 spin tips along with the centrifuge column adaptor into a new 2 ml low bind Eppendorf tubes.
SECTION: Phosphopeptide enrichment using TiO2
68. Apply 200 µL of suspended peptide sample to the spin tip. Centrifuge at 1000 x g, 5 min.

SECTION: Phosphopeptide enrichment using TiO2
69. Reapply sample in the microcentrifuge tube to the spin tip. Centrifuge at 1000 x g, 5 min.
 
 

SECTION: Phosphopeptide enrichment using TiO2
70. Transfer the TiO2 spin tips along with the centrifuge column adaptor into a new 2 ml low bind Eppendorf tubes.
SECTION: Phosphopeptide enrichment using TiO2
71. Wash column by adding 20 µL of Binding/Equilibration Buffer. Centrifuge at 3000 x g, 2 min.
SECTION: Phosphopeptide enrichment using TiO2
72. Wash column by adding 20 µL of Wash Buffer. Centrifuge at 3000 x g, 2 min.

SECTION: Phosphopeptide enrichment using TiO2
73. Repeat steps 71 and 72 in a sequential order.
SECTION: Phosphopeptide enrichment using TiO2
74. Wash column by adding 20 µL of LC-MS grade water. Centrifuge at 3000 x g, 2 min.

SECTION: Phosphopeptide enrichment using TiO2
75. Place TiO2 spin tips into new 2 ml low bind Eppendorf tubes. Add 60 µL of elution buffer and centrifuge at 1000 x g, 1 min.

SECTION: Phosphopeptide enrichment using TiO2
76. Repeat step 75 for a second round of elution. Discard spin tips, vortex samples and centrifuge at 17000 x g, 30 s.
SECTION: Phosphopeptide enrichment using TiO2
77. Take 1% of the sample for Phosphopeptide enrichment verification by MS analysis.
SECTION: Phosphopeptide enrichment using TiO2
78. Take 25 % of the sample as a back-up or for Data Independent Acquisition (DIA)-based MS analysis.
SECTION: Phosphopeptide enrichment using TiO2
79. Snap freeze samples on dry ice and subject them for vacuum dryness using Speed Vac concentrator.
SECTION: Phosphopeptide enrichment using TiO2
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 200 µL of elution buffer and repeat elution two more times for a total of 600 µL of eluates.
SECTION: Phosphopeptide enrichment using TiO2
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.

SECTION: Tandem Mass Tags (TMT) labelling of peptides
82. 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   for 20 min.
SECTION: Tandem Mass Tags (TMT) labelling of peptides
83. Take out TMT kit from -80 °C freezer and equilibrate it to reach Room temperature.

SECTION: Tandem Mass Tags (TMT) labelling of peptides
84. 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. 
 

SECTION: Tandem Mass Tags (TMT) labelling of peptides
85. Transfer dissolved peptides into a 0.5 ml low bind Eppendorf tubes.
SECTION: Tandem Mass Tags (TMT) labelling of peptides
86. Add 20 µL of 10 μg/μL TMT reagent i.e., 200 µg.

SECTION: Tandem Mass Tags (TMT) labelling of peptides
87. Give a gentle vortex and brief spin 2000 x g, 1 min.

SECTION: Tandem Mass Tags (TMT) labelling of peptides
88. Place samples on a Thermomixer and incubate at Room temperature for 120 min with a gentle agitation  .

SECTION: Tandem Mass Tags (TMT) labelling of peptides
89. Add another 50 µL of 50 millimolar (mM) TEABC buffer to make a final 100 µL reaction. Vortex, brief spin at 2000 x g, 1 min and incubate on a Thermomixer for 10 min.  
 

SECTION: Tandem Mass Tags (TMT) labelling of peptides
90. 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. 
 

SECTION: Tandem Mass Tags (TMT) labelling of peptides
91. 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.
SECTION: Tandem Mass Tags (TMT) labelling of peptides
92. Thaw stored TMT labelled samples from step 91 to Room temperature.

SECTION: Tandem Mass Tags (TMT) labelling of peptides
93. Prepare 5% (by vol) final Hydroxyl amine solution by dissolving in water from a 50% (by vol) stock solution.
SECTION: Tandem Mass Tags (TMT) labelling of peptides
94. 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 20 min.

SECTION: Tandem Mass Tags (TMT) labelling of peptides
95. Pool all samples into a single tube.
SECTION: Tandem Mass Tags (TMT) labelling of peptides
96. 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. 
 

SECTION: Tandem Mass Tags (TMT) labelling of peptides
97. Snap freeze the remaining 880 µL reaction and vacuum dry using Speed Vac.
SECTION: Tandem Mass Tags (TMT) labelling of peptides
98. Submit samples to MS facility for high pH fractionation.
SECTION: Tandem Mass Tags (TMT) labelling of peptides
99. Dissolve the digested peptide by adding 120 µL of High-pH Solvent-A (10 millimolar (mM) Ammonium formate pH 10.0). Place the sample on a Thermomixer with an agitation at   for 30 min. Centrifuge at 17000 x g, 5 min.

SECTION: Tandem Mass Tags (TMT) labelling of peptides
100. Verify the pH to be ~ pH 10.0. If pH appears to be low, adjust with Ammonium hydroxide (38% (by vol) by adding 1 µL and recheck the pH.
SECTION: Tandem Mass Tags (TMT) labelling of peptides
101. Ensure the LC-solvents are as Solvent-A (10 millimolar (mM) Ammonium formate pH 10.0); Solvent-B (90% ACN (v/v) in 10 millimolar (mM) Ammonium formate pH 10.0). 
 

SECTION: Tandem Mass Tags (TMT) labelling of peptides
102. Prepare the LC method by following the below gradient:  

 
     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   

SECTION: Tandem Mass Tags (TMT) labelling of peptides
103. Set the fraction collection time as Start time (min) 5.5 and End time (min) 62.0.
SECTION: Tandem Mass Tags (TMT) labelling of peptides
104. Collect a total of 96 fractions by keeping the fraction collection for 1 min for each fraction.

SECTION: Tandem Mass Tags (TMT) labelling of peptides
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.
SECTION: Tandem Mass Tags (TMT) labelling of peptides
106. Snap freeze and vacuum dry using Speed Vac concentrator.
SECTION: Tandem Mass Tags (TMT) labelling of peptides
107. 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.
SECTION: Tandem Mass Tags (TMT) labelling of peptides
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.
SECTION: LC-MS/MS analysis on Orbitrap Lumos Tribrid mass spectrometer for Phosphoproteomic analysis
109. Take 2 µg of each fraction from Phosphoproteomic experiment, transfer into LC vial and place it in LC autosampler tray.
SECTION: LC-MS/MS analysis on Orbitrap Lumos Tribrid mass spectrometer for Phosphoproteomic analysis
110. Construct LC and MS method using the below settings.
SECTION: LC-MS/MS analysis on Orbitrap Lumos Tribrid mass spectrometer for Phosphoproteomic analysis
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). 
 
 
     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   


SECTION: LC-MS/MS analysis on Orbitrap Lumos Tribrid mass spectrometer for Phosphoproteomic analysis
112. Mass spectrometer parameters: Refer below settings to construct FT-FT-HCD (MS2) method: 
     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        
     

SECTION: LC-MS/MS analysis on Orbitrap Lumos Tribrid mass spectrometer for Phosphoproteomic analysis
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.
SECTION: LC-MS/MS analysis on Orbitrap Lumos Tribrid mass spectrometer for total proteomic analysis
114. Take 2 µg of each fraction from Phosphoproteomics experiment, transfer into LC vial and place it in LC autosampler tray.
SECTION: LC-MS/MS analysis on Orbitrap Lumos Tribrid mass spectrometer for total proteomic analysis
115. Construct LC and MS method using the below settings.
SECTION: LC-MS/MS analysis on Orbitrap Lumos Tribrid mass spectrometer for total proteomic analysis
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). 
     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   

SECTION: LC-MS/MS analysis on Orbitrap Lumos Tribrid mass spectrometer for total proteomic analysis
117. Mass spectrometer parameters: Refer below settings to construct FT-IT-HCD-FT-HCD (MS3) method: 
     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   

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