Result name: 2019-03-06_CRmice_TMT10_input_AkshayBareja_2019-02-14_Run Description: Processing workflow for reporter ion-based quantification. HCD spectra using SequestHTwith Percolator validation. Specify the FASTA database, label used, and any additional modifications. Workflow based on template: PD_22_QE_Mouse_Input_TMT Creation date: 03/22/2019 14:01:51 ------------------------------------------------------------------ The workflow tree: ------------------------------------------------------------------ |-(19) Spectrum Files |-(4) Reporter Ions Quantifier |-(20) Spectrum Selector |-(8) MS Amanda 2.0 |-(12) Percolator |-(14) Sequest HT |-(9) Percolator ------------------------------------------------------------------ Processing node 19: Spectrum Files ------------------------------------------------------------------ Input Data: - File Name(s) (Hidden): W:\Lab.Equipment\Q.Exactive\Proteomics\2019-03-06_JWhite_AkshayBareja_CaloricRestriction-TMT10_Phos+Input\Input\2019-03-06_CRmice_TMT10_input_AkshayBareja_2019-02-14_Run1.raw W:\Lab.Equipment\Q.Exactive\Proteomics\2019-03-06_JWhite_AkshayBareja_CaloricRestriction-TMT10_Phos+Input\Input\2019-03-06_CRmice_TMT10_input_AkshayBareja_2019-02-14_Run2.raw W:\Lab.Equipment\Q.Exactive\Proteomics\2019-03-06_JWhite_AkshayBareja_CaloricRestriction-TMT10_Phos+Input\Input\2019-03-06_CRmice_TMT10_input_AkshayBareja_2019-02-14_Run3.raw ------------------------------------------------------------------ Processing node 4: Reporter Ions Quantifier ------------------------------------------------------------------ 0. Quantification Method: - Quantification Method: TMT10plex_ave_60_percent_all_kits 1. Peak Integration: - Integration Tolerance: 10 ppm - Integration Method: Most Confident Centroid 2. Scan Event Filters: - Mass Analyzer: FTMS - MS Order: MS2 - Activation Type: HCD - Min. Collision Energy: 0 - Max. Collision Energy: 1000 ------------------------------------------------------------------ Processing node 20: Spectrum Selector ------------------------------------------------------------------ 1. General Settings: - Precursor Selection: Use MS1 Precursor - Use Isotope Pattern in Precursor Reevaluation: True - Provide Profile Spectra: Automatic 2. Spectrum Properties Filter: - Lower RT Limit: 0 - Upper RT Limit: 0 - First Scan: 0 - Last Scan: 0 - Lowest Charge State: 0 - Highest Charge State: 0 - Min. Precursor Mass: 350 Da - Max. Precursor Mass: 5000 Da - Total Intensity Threshold: 0 - Minimum Peak Count: 1 3. Scan Event Filters: - MS Order: Is Not MS1 - Min. Collision Energy: 0 - Max. Collision Energy: 1000 - Scan Type: Is Full 4. Peak Filters: - S/N Threshold (FT-only): 1.5 5. Replacements for Unrecognized Properties: - Unrecognized Charge Replacements: Automatic - Unrecognized Mass Analyzer Replacements: ITMS - Unrecognized MS Order Replacements: MS2 - Unrecognized Activation Type Replacements: CID - Unrecognized Polarity Replacements: + - Unrecognized MS Resolution@200 Replacements: 60000 - Unrecognized MSn Resolution@200 Replacements: 30000 6. Precursor Pattern Extraction: - Precursor Clipping Range Before: 2.5 Da - Precursor Clipping Range After: 5.5 Da ------------------------------------------------------------------ Processing node 8: MS Amanda 2.0 ------------------------------------------------------------------ 1. Input Data: - Protein Database: Mouse_UP000000589_20190306.fasta - Enzyme Name: Trypsin (Full) - Missed Cleavages: 2 - MS1 tolerance: 10 ppm - MS2 tolerance: 0.02 Da 2. Static Modifications: - 2. Static Modification: Carbamidomethyl / +57.021 Da (C) - 3. Static Modification: TMT6plex / +229.163 Da (K) - Static Peptide N-Terminal Modification: TMT6plex / +229.163 Da (N-Terminus) 3. Dynamic Modifications: - 2. Dynamic Modification: Oxidation / +15.995 Da (M) 4. Additional Settings: - Max No. of same modifs: 3 - Max No. of dynamic modifs: 4 - Max number of same neutral losses (H2O, NH3): 1 - No. of considered NLs (modifications): 2 - Max. No. modif sites: 6 - Ion Settings: b,y - Max. Rank: 5 - Peptide Cut Off Score: 1 - Minimum Peptide Length: 0 - Perform deisotoping: True - Use monoisotopic mass: True 5. Performance Settings: - Protein Database Size: 200000 - Number of spectra per package: 10000 ------------------------------------------------------------------ Processing node 12: Percolator ------------------------------------------------------------------ 1. Target/Decoy Strategy: - Target/Decoy Selection: Concatenated - Validation based on: q-Value 2. Input Data: - Maximum Delta Cn: 0.05 - Maximum Rank: 0 3. FDR Targets: - Target FDR (Strict): 0.01 - Target FDR (Relaxed): 0.05 ------------------------------------------------------------------ Processing node 14: Sequest HT ------------------------------------------------------------------ 1. Input Data: - Protein Database: Mouse_UP000000589_20190306.fasta - Enzyme Name: Trypsin (Full) - Max. Missed Cleavage Sites: 2 - Min. Peptide Length: 6 - Max. Peptide Length: 144 - Max. Number of Peptides Reported: 10 2. Tolerances: - Precursor Mass Tolerance: 10 ppm - Fragment Mass Tolerance: 0.02 Da - Use Average Precursor Mass: False - Use Average Fragment Mass: False 3. Spectrum Matching: - Use Neutral Loss a Ions: True - Use Neutral Loss b Ions: True - Use Neutral Loss y Ions: True - Use Flanking Ions: True - Weight of a Ions: 0 - Weight of b Ions: 1 - Weight of c Ions: 0 - Weight of x Ions: 0 - Weight of y Ions: 1 - Weight of z Ions: 0 4. Dynamic Modifications: - Max. Equal Modifications Per Peptide: 3 - Max. Dynamic Modifications Per Peptide: 4 - 2. Dynamic Modification: Oxidation / +15.995 Da (M) 7. Static Modifications: - Peptide N-Terminus: TMT6plex / +229.163 Da (Any N-Terminus) - 1. Static Modification: Carbamidomethyl / +57.021 Da (C) - 2. Static Modification: TMT6plex / +229.163 Da (K) ------------------------------------------------------------------ Processing node 9: Percolator ------------------------------------------------------------------ 1. Target/Decoy Strategy: - Target/Decoy Selection: Concatenated - Validation based on: q-Value 2. Input Data: - Maximum Delta Cn: 0.05 - Maximum Rank: 0 3. FDR Targets: - Target FDR (Strict): 0.01 - Target FDR (Relaxed): 0.05

Result name: 2019-03-06_CRmice_TMT10_phos_AkshayBareja_2019-02-20 Description: Processing workflow for reporter ion-based quantification. HCD spectra using SequestHTwith Percolator validation. Specify the FASTA database, label used, and any additional modifications. Workflow based on template: PD_23_QE_Mouse_Phospho_TMT_SeaquestHT_MSA20 Creation date: 03/12/2019 15:00:49 ------------------------------------------------------------------ The workflow tree: ------------------------------------------------------------------ |-(15) Spectrum Files |-(4) Reporter Ions Quantifier |-(16) Spectrum Selector |-(8) MS Amanda 2.0 |-(12) Percolator |-(7) IMP-ptmRS |-(14) Sequest HT |-(9) Percolator |-(7) IMP-ptmRS ------------------------------------------------------------------ Processing node 15: Spectrum Files ------------------------------------------------------------------ Input Data: - File Name(s) (Hidden): W:\Lab.Equipment\Q.Exactive\Proteomics\2019-03-06_JWhite_AkshayBareja_CaloricRestriction-TMT10_Phos+Input\Phospho\2019-03-06_CRmice_TMT10_phos_AkshayBareja_2019-02-20.raw W:\Lab.Equipment\Q.Exactive\Proteomics\2019-03-06_JWhite_AkshayBareja_CaloricRestriction-TMT10_Phos+Input\Phospho\2019-03-06_CRmice_TMT10_phos_AkshayBareja_2019-02-20_Run2.raw W:\Lab.Equipment\Q.Exactive\Proteomics\2019-03-06_JWhite_AkshayBareja_CaloricRestriction-TMT10_Phos+Input\Phospho\2019-03-06_CRmice_TMT10_phos_AkshayBareja_2019-02-20_Run3.raw ------------------------------------------------------------------ Processing node 4: Reporter Ions Quantifier ------------------------------------------------------------------ 0. Quantification Method: - Quantification Method: TMT10plex_ave_60_percent_all_kits 1. Peak Integration: - Integration Tolerance: 10 ppm - Integration Method: Most Confident Centroid 2. Scan Event Filters: - Mass Analyzer: FTMS - MS Order: MS2 - Activation Type: HCD - Min. Collision Energy: 0 - Max. Collision Energy: 1000 ------------------------------------------------------------------ Processing node 16: Spectrum Selector ------------------------------------------------------------------ 1. General Settings: - Precursor Selection: Use MS1 Precursor - Use Isotope Pattern in Precursor Reevaluation: True - Provide Profile Spectra: Automatic 2. Spectrum Properties Filter: - Lower RT Limit: 0 - Upper RT Limit: 0 - First Scan: 0 - Last Scan: 0 - Lowest Charge State: 0 - Highest Charge State: 0 - Min. Precursor Mass: 350 Da - Max. Precursor Mass: 5000 Da - Total Intensity Threshold: 0 - Minimum Peak Count: 1 3. Scan Event Filters: - MS Order: Is Not MS1 - Min. Collision Energy: 0 - Max. Collision Energy: 1000 - Scan Type: Is Full 4. Peak Filters: - S/N Threshold (FT-only): 1.5 5. Replacements for Unrecognized Properties: - Unrecognized Charge Replacements: Automatic - Unrecognized Mass Analyzer Replacements: ITMS - Unrecognized MS Order Replacements: MS2 - Unrecognized Activation Type Replacements: CID - Unrecognized Polarity Replacements: + - Unrecognized MS Resolution@200 Replacements: 60000 - Unrecognized MSn Resolution@200 Replacements: 30000 6. Precursor Pattern Extraction: - Precursor Clipping Range Before: 2.5 Da - Precursor Clipping Range After: 5.5 Da ------------------------------------------------------------------ Processing node 8: MS Amanda 2.0 ------------------------------------------------------------------ 1. Input Data: - Protein Database: Mouse_UP000000589_20190306.fasta - Enzyme Name: Trypsin (Full) - Missed Cleavages: 2 - MS1 tolerance: 10 ppm - MS2 tolerance: 0.02 Da 2. Static Modifications: - 1. Static Modification: TMT6plex / +229.163 Da (K) - 2. Static Modification: Carbamidomethyl / +57.021 Da (C) - Static Peptide N-Terminal Modification: TMT6plex / +229.163 Da (N-Terminus) 3. Dynamic Modifications: - 2. Dynamic Modification: Oxidation / +15.995 Da (M) - 3. Dynamic Modification: Phospho / +79.966 Da (S, T, Y) 4. Additional Settings: - Max No. of same modifs: 3 - Max No. of dynamic modifs: 4 - Max number of same neutral losses (H2O, NH3): 1 - No. of considered NLs (modifications): 2 - Max. No. modif sites: 6 - Ion Settings: b,y - Max. Rank: 5 - Peptide Cut Off Score: 1 - Minimum Peptide Length: 0 - Perform deisotoping: True - Use monoisotopic mass: True 5. Performance Settings: - Protein Database Size: 200000 - Number of spectra per package: 10000 ------------------------------------------------------------------ Processing node 12: Percolator ------------------------------------------------------------------ 1. Target/Decoy Strategy: - Target/Decoy Selection: Concatenated - Validation based on: q-Value 2. Input Data: - Maximum Delta Cn: 0.05 - Maximum Rank: 0 3. FDR Targets: - Target FDR (Strict): 0.01 - Target FDR (Relaxed): 0.05 ------------------------------------------------------------------ Processing node 7: IMP-ptmRS ------------------------------------------------------------------ 1. Scoring: - PhosphoRS Mode: True - Report Only PTMs: True - Use Diagnostic Ions: True - Use Fragment Mass Tolerance of Search Node: True - Fragment Mass Tolerance: 0.5 Da - Consider neutral loss peaks for CID, HCD and EThcD: Automatic - Maximum Peak Depth: 8 - Use a mass accuracy correction: False 2. Performance: - Maximum Number of Position Isoforms: 500 - Maximum PTMs per peptide: 10 ------------------------------------------------------------------ Processing node 14: Sequest HT ------------------------------------------------------------------ 1. Input Data: - Protein Database: Mouse_UP000000589_20190306.fasta - Enzyme Name: Trypsin (Full) - Max. Missed Cleavage Sites: 2 - Min. Peptide Length: 6 - Max. Peptide Length: 144 - Max. Number of Peptides Reported: 10 2. Tolerances: - Precursor Mass Tolerance: 10 ppm - Fragment Mass Tolerance: 0.02 Da - Use Average Precursor Mass: False - Use Average Fragment Mass: False 3. Spectrum Matching: - Use Neutral Loss a Ions: True - Use Neutral Loss b Ions: True - Use Neutral Loss y Ions: True - Use Flanking Ions: True - Weight of a Ions: 0 - Weight of b Ions: 1 - Weight of c Ions: 0 - Weight of x Ions: 0 - Weight of y Ions: 1 - Weight of z Ions: 0 4. Dynamic Modifications: - Max. Equal Modifications Per Peptide: 3 - Max. Dynamic Modifications Per Peptide: 4 - 2. Dynamic Modification: Oxidation / +15.995 Da (M) - 3. Dynamic Modification: Phospho / +79.966 Da (S, T, Y) 7. Static Modifications: - Peptide N-Terminus: TMT6plex / +229.163 Da (Any N-Terminus) - 1. Static Modification: Carbamidomethyl / +57.021 Da (C) - 3. Static Modification: TMT6plex / +229.163 Da (K) ------------------------------------------------------------------ Processing node 9: Percolator ------------------------------------------------------------------ 1. Target/Decoy Strategy: - Target/Decoy Selection: Concatenated - Validation based on: q-Value 2. Input Data: - Maximum Delta Cn: 0.05 - Maximum Rank: 0 3. FDR Targets: - Target FDR (Strict): 0.01 - Target FDR (Relaxed): 0.05