TITLE: GFP Immunoprecipitation and Sample Preparation for Tandem Mass Tag (TMT) Mass Spectrometry Analysis
AUTHORS: Prosenjit Pal, Raja S. Nirujogi, Francesca Tonelli, Dario R Alessi

[DESCRIPTION]
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. 

[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.


[STEPS]
SECTION: Transient transfection of HEK293 cells
1. 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).
 

SECTION: Transient transfection of HEK293 cells
2. Prepare a transfection mix in a sterile 1.5ml Eppendorf tube, containing (for each 10 cm dish):
 
      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  
 
SECTION: Transient transfection of HEK293 cells
3. Mix by vortexing and incubate at Room temperature for 20 min.

SECTION: Transient transfection of HEK293 cells
4. Add the mixture dropwise to the cells from step 1 using a P1000 sterile pipette.
SECTION: Transient transfection of HEK293 cells
5. Incubate cells at 37 °C for 1440 min.

SECTION: Preparation and quantification of cell lysates
6. Remove culture medium completely from each dish using an aspirator.
SECTION: Preparation and quantification of cell lysates
7. Quickly rinse cells in the tissue culture dish by carefully pouring culture media without Foetal bovine serum (at Room temperature) into the dish. 
 


SECTION: Preparation and quantification of cell lysates
8. Pour off media from the culture dish and completely aspirate any residual media.
SECTION: Preparation and quantification of cell lysates
9. Immediately add 400 µL of ice-cold complete lysis buffer to each dish ensuring that the entire surface is covered by lysis buffer.

SECTION: Preparation and quantification of cell lysates
10. Transfer the plate on ice.

SECTION: Preparation and quantification of cell lysates
11. Scrape the cells on the dish using a cell lifter to ensure all cells are detached from the dish.
SECTION: Preparation and quantification of cell lysates
12. Using a pipette, transfer the lysate to a 1.5mL Eppendorf tube.
SECTION: Preparation and quantification of cell lysates
13. Leave samples on ice for 20-30 minutes to allow for efficient lysis.

SECTION: Preparation and quantification of cell lysates
14. Spin down lysates at 17000 x g, 10 min, 4 °C.

SECTION: Preparation and quantification of cell lysates
15. Transfer supernatant to a new Eppendorf tube and discard the pellet.
SECTION: Preparation and quantification of cell lysates
16. Proceed to estimating the protein concentration of cell lysates by Bradford assay according to the manufacturer’s instructions. 
 

SECTION: Immunoprecipitation of GFP-tagged proteins from cell lysates
17. Transfer n x 20 µL of packed ChromoTek GFP-Trap Agarose Beads (where n = number of samples) into a low binding Eppendorf tube. 

SECTION: Immunoprecipitation of GFP-tagged proteins from cell lysates
18. Pellet the beads by centrifuging at 2500 x g, 3 min, 4 °C.

SECTION: Immunoprecipitation of GFP-tagged proteins from cell lysates
19. Carefully aspirate the supernatant.
SECTION: Immunoprecipitation of GFP-tagged proteins from cell lysates
20. Resuspend the beads in 1 mL of IP wash buffer.

SECTION: Immunoprecipitation of GFP-tagged proteins from cell lysates
21. Repeat steps 18 to 20 twice.
SECTION: Immunoprecipitation of GFP-tagged proteins from cell lysates
22. Centrifuge at 2500 x g, 3 min, 4 °C and aspirate the supernatant.

SECTION: Immunoprecipitation of GFP-tagged proteins from cell lysates
23. 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.

SECTION: Immunoprecipitation of GFP-tagged proteins from cell lysates
24. 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.
SECTION: Immunoprecipitation of GFP-tagged proteins from cell lysates
25. For each sample, transfer 500 µg lysate from step 15 to the washed beads.

SECTION: Immunoprecipitation of GFP-tagged proteins from cell lysates
26. Incubate for 120 min at 4 °C under mild agitation (on an orbital shaker).

SECTION: Immunoprecipitation of GFP-tagged proteins from cell lysates
27. Pellet the beads by centrifuging at 2500 x g, 5 min, 4 °C.

SECTION: Immunoprecipitation of GFP-tagged proteins from cell lysates
28. Carefully aspirate the supernatant.
SECTION: Immunoprecipitation of GFP-tagged proteins from cell lysates
29. Resuspend the beads in 1 mL of IP wash buffer.

SECTION: Immunoprecipitation of GFP-tagged proteins from cell lysates
30. Repeat steps 27 to 29 twice.
SECTION: Immunoprecipitation of GFP-tagged proteins from cell lysates
31. Centrifuge at 2500 x g, 5 min, 4 °C and aspirate the supernatant.

SECTION: Immunoprecipitation of GFP-tagged proteins from cell lysates
32. Immediately proceed to Elution and on-bead tryptic digestion of immunoprecipitated proteins .
SECTION: Elution and on-bead tryptic digestion of immunoprecipitated proteins
33. Add 100 µL of elution buffer I to the beads from step 32.

SECTION: Elution and on-bead tryptic digestion of immunoprecipitated proteins
34. Add 500 ng of sequencing grade trypsin to the mixture and incubate on a Thermomixer at  .

SECTION: Elution and on-bead tryptic digestion of immunoprecipitated proteins
35. Centrifuge the mixture at 2500 x g, 2 min. Carefully transfer the supernatant to new Eppendorf tubes, being careful not to disturb the beads. 

SECTION: Elution and on-bead tryptic digestion of immunoprecipitated proteins
36. 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, 2 min. Carefully transfer the supernatant to the collection Eppendorf tubes from step 35, being careful not to disturb the beads. 
SECTION: Elution and on-bead tryptic digestion of immunoprecipitated proteins
37. Incubate the Eppendorf tubes on the Thermomixer    120 min (or minimum of 12 hr).
SECTION: Elution and on-bead tryptic digestion of immunoprecipitated proteins
38. Add 1% (v/v) Trifluoroacetic Acid (TFA) to the digested peptides from step 37. Incubate for 5 min at Room temperature and centrifuge at 17000 x g, 10 min.

SECTION: Peptide clean-up using C18 stage-tips
39. Prepare a C18 stage-tip for each sample as described in dx.doi.org/10.17504/protocols.io.bs3tngnn. 
 

SECTION: Peptide clean-up using C18 stage-tips
40. C18 stage tips activation: add 80 µL of 100% ACN to each stage-tip and centrifuge at 2000 x g, 1 min.

SECTION: Peptide clean-up using C18 stage-tips
41. C18 stage tips equilibration: add 80 µL of 0.1% TFA to each stage-tip and centrifuge at 2000 x g, 2 min.

SECTION: Peptide clean-up using C18 stage-tips
42. Repeat step 41.
SECTION: Peptide clean-up using C18 stage-tips
43. Transfer the C18 stage-tip to a new low-binding Eppendorf.
SECTION: Peptide clean-up using C18 stage-tips
44. Load the digested peptides from step 38 onto the C18 stage-tip from step 43 and centrifuge at 2000 x g, 4 min.

SECTION: Peptide clean-up using C18 stage-tips
45. Collect the flowthrough from step 44 and re-load onto the same C18 stage-tip. Centrifuge at 2000 x g, 4 min.

SECTION: Peptide clean-up using C18 stage-tips
46. Wash the C18 stage-tips by adding 80 µL of 0.1% TFA and centrifuging at 2000 x g, 2 min.

SECTION: Peptide clean-up using C18 stage-tips
47. Repeat step 46.
SECTION: Peptide clean-up using C18 stage-tips
48. Transfer the C18 stage-tip to a new low-binding Eppendorf.
SECTION: Peptide clean-up using C18 stage-tips
49. Add 30 µL of 30% (v/v) ACN in 0.1% (v/v) TFA to each stage-tip and centrifuge at 1000 x g, 1 min.

SECTION: Peptide clean-up using C18 stage-tips
50. Repeat step 49.
SECTION: Peptide clean-up using C18 stage-tips
51. Take 1-2 µL of the digested peptides, vacuum dry and inject on MS to verify the digestion efficiency.
 


SECTION: Peptide clean-up using C18 stage-tips
52. Vacuum dry completely the remaining peptides and store at -80 °C until ready to undertake TMT labelling.

SECTION: Tandem Mass Tag Labelling
53. 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.

SECTION: Tandem Mass Tag Labelling
54. Leave at Room temperature for 10 min, then vortex and spin 2000 x g, 2 min.
 




SECTION: Tandem Mass Tag Labelling
55. 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. 
 

SECTION: Tandem Mass Tag Labelling
56. Place the samples in a water bath sonicator for 10 min.

SECTION: Tandem Mass Tag Labelling
57. Centrifuge the samples  17000 x g, 10 min.

SECTION: Tandem Mass Tag Labelling
58. Transfer dissolved peptides into a 1.5ml protein low binding Eppendorf tube.
SECTION: Tandem Mass Tag Labelling
59. Add 10 µL 20 μg/μL TMT reagent i.e. 200 µg aiming for a 1:1 mass ratio of peptide: TMT reagent.

SECTION: Tandem Mass Tag Labelling
60. Give a gentle vortex and spin at 2000 x g, 1 min.

SECTION: Tandem Mass Tag Labelling
61. Place samples on a Thermomixer and incubate with gentle agitation at   .

SECTION: Tandem Mass Tag Labelling
62. Add another 50 µL 50 millimolar (mM) TEAB buffer to make a final 100 µL reaction. Vortex, brief spin at 2000 x g, 10 min and incubate on a Thermomixer for 10 min. 
 

SECTION: Tandem Mass Tag Labelling
63. 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. 
 


SECTION: Tandem Mass Tag Labelling
64. 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 Tag Labelling
65. Thaw stored TMT labelled samples from step 63 to Room temperature.

SECTION: Tandem Mass Tag Labelling
66. Prepare 5% (by vol) final Hydroxyl amine solution by dissolving in water from a 50% (by vol) stock solution.
SECTION: Tandem Mass Tag Labelling
67. 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 20 min.

SECTION: Tandem Mass Tag Labelling
68. Pool all samples into a single tube.
SECTION: Tandem Mass Tag Labelling
69. Transfer 20% of the reaction to a new low-binding Eppendorf tube as a backup: snap freeze on dry ice and vacuum dry. 
 


SECTION: Tandem Mass Tag Labelling
70. Snap freeze the remaining 80% of the reaction and vacuum dry using Speed vac.

SECTION: Mini-basic RPLC fractionation
71. 
 
Prepare four C18 stage-tips as described in dx.doi.org/10.17504/protocols.io.bs3tngnn.
SECTION: Mini-basic RPLC fractionation
72. Label eight 1.5ml low-binding Eppendorf tubes as "Fraction 1" to "Fraction 8".
SECTION: Mini-basic RPLC fractionation
73. Prepare 50 mL of bRP stock solution (50 millimolar (mM) Ammonium formate in Milli-Q H2O).

SECTION: Mini-basic RPLC fractionation
74. 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).

SECTION: Mini-basic RPLC fractionation
75. Prepare Solvent B: Mix 20 mL of bRP stock solution with 20 mL of 100% Acetonitrile (=25 millimolar (mM) Ammonium formate in 50% ACN).

SECTION: Mini-basic RPLC fractionation
76. 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. 
     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   
 
SECTION: Mini-basic RPLC fractionation
77. Dissolve peptides from step 58 in 200 µL of Solvent A. 
 

SECTION: Mini-basic RPLC fractionation
78. Place samples on a Thermomixer at  .

SECTION: Mini-basic RPLC fractionation
79. Centrifuge sample at 17000 x g, 5 min.

SECTION: Mini-basic RPLC fractionation
80. Transfer the supernatant into a new 1.5ml protein low-binding Eppendorf tube.
SECTION: Mini-basic RPLC fractionation
81. Add 200 µL of 100% ACN to the C18 stage-tips (Step 71) and centrifuge at 2500 x g, 2 min to activate the columns. Discard the flow through.

SECTION: Mini-basic RPLC fractionation
82. Add 200 µL of Solvent B to each column from step 81 and centrifuge at 2500 x g, 2 min. Discard the flow through.
SECTION: Mini-basic RPLC fractionation
83. Add 200 µL of Solvent A to each column from step 82 and centrifuge at 2500 x g, 2 min .

SECTION: Mini-basic RPLC fractionation
84. Transfer the column to a new low-binding Eppendorf tube.
SECTION: Mini-basic RPLC fractionation
85. Sample loading: Slowly load each sample (from step 80) onto a column (from step 84).
SECTION: Mini-basic RPLC fractionation
86. Centrifuge at 1500 x g, 5 min.

SECTION: Mini-basic RPLC fractionation
87. Collect the flowthrough from step 86 and slowly load onto the same column.
SECTION: Mini-basic RPLC fractionation
88. Centrifuge at  1500 x g, 5 min.
SECTION: Mini-basic RPLC fractionation
89. Transfer the column into a new 1.5ml Eppendorf tube.
SECTION: Mini-basic RPLC fractionation
90. To wash the column, add 200 µL of Solvent A to the column and centrifuge at 2500 x g, 2 min.

SECTION: Mini-basic RPLC fractionation
91. Transfer the column into the tube labelled as “Fraction 1” (from step 72).
SECTION: Mini-basic RPLC fractionation
92. Add 60 µL of Elution solvent 1 (from step 76) to the column and centrifuge at 1500 x g, 2 min.

SECTION: Mini-basic RPLC fractionation
93. 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, 2 min.

SECTION: Mini-basic RPLC fractionation
94. 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.
SECTION: Mini-basic RPLC fractionation
95. Place fractions on dry ice and vacuum dry completely using a SpeedVac.
SECTION: LC-MS/MS analysis
96. Dissolve each fraction from step 95 in 20 µL of LC-buffer (3% (v/v) ACN, 0.1% (v/v) formic acid).
SECTION: LC-MS/MS analysis
97. Place samples on a Thermomixer at  .

SECTION: LC-MS/MS analysis
98. 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.

SECTION: LC-MS/MS analysis
99. 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.
 
     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   
 
SECTION: LC-MS/MS analysis
100. The raw data was searched using MaxQuant version 1.6.6.0 using the parameters described below.
 
     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