O-Pair

[peteythecat1]

Does O-Pair work for current generation of Orbitrap instruments (Eclipse, Ascend, Excedion Pro) where immediate HCD-pd-EThcD adjacency is no longer guaranteed because of how the real-time parallelization and Orbitrap scheduling work. For example HCD and EThcD can be several scans apart. Some times 7 scans apart.

[dpolasky]

Hi @peteythecat1,
There is no maximum number of intervening MS2 scans for the scan pairing, the only requirement is that the paired scans are within the same "cycle" before the next MS1 scan. As long as that's the case, it should have no issue.

Best,
Dan

[peteythecat1]

This was an issue on Metamorpheus version of O-pair. So not an issue on the feature within Fragger then?

[dpolasky]

Correct - the scan pairing is handled separately by FragPipe (i.e., it is not carried over from Metamorpheus), and I guess the method is different.

Best,
Dan

[peteythecat1]

Hi Dan, Is their Tutorial to run O-pair on fragger. I am familiar with O-pair output on Metamorpheus but it seems quite different on Fragger. One additional question, is there support for EThcd , similar to O-pair on Metamorpheus? From: Daniel Polasky ***@***.***> Sent: Wednesday, October 29, 2025 11:32 AM To: Nesvilab/FragPipe ***@***.***> Cc: Saba, Julian ***@***.***>; Mention ***@***.***> Subject: Re: [Nesvilab/FragPipe] O-Pair (Issue #2522) CAUTION: This email originated from outside of Thermo Fisher Scientific. If you believe it to be suspicious, report using the 'Report this email' button, which is displayed with a shield icon in Outlook, or send to ***@***.*** [https://avatars.githubusercontent.com/u/15036076?s=20&v=4]dpolasky left a comment (Nesvilab/FragPipe#2522)<https://urldefense.com/v3/__https:/github.com/Nesvilab/FragPipe/issues/2522*issuecomment-3462521103__;Iw!!HLrAl2XzZ3iCLg!FWg6BUWwxV2JG1S4HwHMiYLgET4-pHxr3IuAFq9T1LBgKVdcaGwa3FRuroI3VZNpGWNSLlQ06hqRssfLEMTKtrbK9ma4hdI$> Hi @peteythecat1<https://urldefense.com/v3/__https:/github.com/peteythecat1__;!!HLrAl2XzZ3iCLg!FWg6BUWwxV2JG1S4HwHMiYLgET4-pHxr3IuAFq9T1LBgKVdcaGwa3FRuroI3VZNpGWNSLlQ06hqRssfLEMTKtrbKqnT17bQ$>, There is no maximum number of intervening MS2 scans for the scan pairing, the only requirement is that the paired scans are within the same "cycle" before the next MS1 scan. As long as that's the case, it should have no issue. Best, Dan — Reply to this email directly, view it on GitHub<https://urldefense.com/v3/__https:/github.com/Nesvilab/FragPipe/issues/2522*issuecomment-3462521103__;Iw!!HLrAl2XzZ3iCLg!FWg6BUWwxV2JG1S4HwHMiYLgET4-pHxr3IuAFq9T1LBgKVdcaGwa3FRuroI3VZNpGWNSLlQ06hqRssfLEMTKtrbK9ma4hdI$>, or unsubscribe<https://urldefense.com/v3/__https:/github.com/notifications/unsubscribe-auth/BQBE3DFRLHLVDO2MHANHAGL32DTYVAVCNFSM6AAAAACKSG24SOVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMZTINRSGUZDCMJQGM__;!!HLrAl2XzZ3iCLg!FWg6BUWwxV2JG1S4HwHMiYLgET4-pHxr3IuAFq9T1LBgKVdcaGwa3FRuroI3VZNpGWNSLlQ06hqRssfLEMTKtrbKWzWe2zA$>. You are receiving this because you were mentioned.Message ID: ***@***.******@***.***>>

[dpolasky]

Yes, and yes - you can find the tutorial here: https://fragpipe.nesvilab.org/docs/tutorial_glyco.html#o-glycan-localization-with-o-pair

EThcD is supported both as the paired scan and standalone - if doing standalone EThcD, the MSFragger settings need to be adjusted as well, so it's best to load the glyco-O-hybrid workflow as a starting point.

Best,
Dan

[peteythecat1]

Hi Dan, I have HCD-pd-EThcD O-GlcNAc glycopeptide data acquired on the new Orbitrap Excedion Pro mass spectrometer (It was launched at ASMS this year). I can use O-pair within Metamorpheus to get ID. Whether run as pair or just searching EThcD data. However, I am struggling to get any IDs within O-pair in Fragger. Whether run as pair or stand alone EThcD search. I’ve tried both glyco-hybrid and glyco-O-pair. Would it be possible that Fragger doesn’t support new instrument like Orbitrap Excedion Pro? I’ve searched both RAW file and file converted to MZML Julian Saba, Ph.D. Thermo Fisher Scientific Omics Marketing email: ***@***.******@***.***> ***@***.***<https://www.thermofisher.com/ca/en/home/industrial/mass-spectrometry/liquid-chromatography-mass-spectrometry-lc-ms/lc-ms-systems/orbitrap-lc-ms/orbitrap-astral-zoom-mass-spectrometer.html> ***@***.***<https://www.thermofisher.com/ca/en/home/industrial/mass-spectrometry/liquid-chromatography-mass-spectrometry-lc-ms/lc-ms-systems/orbitrap-lc-ms/orbitrap-excedion-pro-mass-spectrometer.html> From: Daniel Polasky ***@***.***> Sent: Friday, October 31, 2025 8:17 AM To: Nesvilab/FragPipe ***@***.***> Cc: Saba, Julian ***@***.***>; Mention ***@***.***> Subject: Re: [Nesvilab/FragPipe] O-Pair (Issue #2522) CAUTION: This email originated from outside of Thermo Fisher Scientific. If you believe it to be suspicious, report using the 'Report this email' button, which is displayed with a shield icon in Outlook, or send to ***@***.*** [https://avatars.githubusercontent.com/u/15036076?s=20&v=4]dpolasky left a comment (Nesvilab/FragPipe#2522)<https://urldefense.com/v3/__https:/github.com/Nesvilab/FragPipe/issues/2522*issuecomment-3473052070__;Iw!!HLrAl2XzZ3iCLg!BLHmX71UiESrRf-0ObIa996u13ETseP64kl8e7n0WEpOFAbdhohqNtXSVgKZs9pvs1av17VwVSCz0ZR5eimMxdq5pR_y9LI$> Yes, and yes - you can find the tutorial here: https://fragpipe.nesvilab.org/docs/tutorial_glyco.html#o-glycan-localization-with-o-pair<https://urldefense.com/v3/__https:/fragpipe.nesvilab.org/docs/tutorial_glyco.html*o-glycan-localization-with-o-pair__;Iw!!HLrAl2XzZ3iCLg!BLHmX71UiESrRf-0ObIa996u13ETseP64kl8e7n0WEpOFAbdhohqNtXSVgKZs9pvs1av17VwVSCz0ZR5eimMxdq5QWz0-7k$> EThcD is supported both as the paired scan and standalone - if doing standalone EThcD, the MSFragger settings need to be adjusted as well, so it's best to load the glyco-O-hybrid workflow as a starting point. Best, Dan — Reply to this email directly, view it on GitHub<https://urldefense.com/v3/__https:/github.com/Nesvilab/FragPipe/issues/2522*issuecomment-3473052070__;Iw!!HLrAl2XzZ3iCLg!BLHmX71UiESrRf-0ObIa996u13ETseP64kl8e7n0WEpOFAbdhohqNtXSVgKZs9pvs1av17VwVSCz0ZR5eimMxdq5pR_y9LI$>, or unsubscribe<https://urldefense.com/v3/__https:/github.com/notifications/unsubscribe-auth/BQBE3DH4YBO7AT4QJCN4L2D32NON7AVCNFSM6AAAAACKSG24SOVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMZTINZTGA2TEMBXGA__;!!HLrAl2XzZ3iCLg!BLHmX71UiESrRf-0ObIa996u13ETseP64kl8e7n0WEpOFAbdhohqNtXSVgKZs9pvs1av17VwVSCz0ZR5eimMxdq50gPYpkE$>. You are receiving this because you were mentioned.Message ID: ***@***.******@***.***>>

[dpolasky]

Hi Julian,
Can you attach the log files from those searches? I don't know any reason Excedion data would not work, but it's always possible that the data format changes with a new instrument in a way we need to explicitly support, but we're happy to do so if that's the case.

Best,
Dan

[peteythecat1]

Hi Dan, The file exists in 53 is run as pair search and the one ends in 37 is run to search EthcD. Wondering if I did something stupid to filter the search? [cid:cee7e551-3609-4b2c-a94c-21be8b54b7a3]<https://www.thermofisher.com/ca/en/home/industrial/mass-spectrometry/liquid-chromatography-mass-spectrometry-lc-ms/lc-ms-systems/orbitrap-lc-ms/orbitrap-astral-zoom-mass-spectrometer.html> [cid:6342e95d-869a-41a1-9825-7d8be55f3084]<https://www.thermofisher.com/ca/en/home/industrial/mass-spectrometry/liquid-chromatography-mass-spectrometry-lc-ms/lc-ms-systems/orbitrap-lc-ms/orbitrap-excedion-pro-mass-spectrometer.html>

________________________________ From: Daniel Polasky ***@***.***> Sent: October 31, 2025 9:45 AM To: Nesvilab/FragPipe ***@***.***> Cc: Saba, Julian ***@***.***>; Mention ***@***.***> Subject: Re: [Nesvilab/FragPipe] O-Pair (Issue #2522) CAUTION: This email originated from outside of Thermo Fisher Scientific. If you believe it to be suspicious, report using the 'Report this email' button, which is displayed with a shield icon in Outlook, or send to ***@***.*** [https://avatars.githubusercontent.com/u/15036076?s=20&v=4]dpolasky left a comment (Nesvilab/FragPipe#2522)<https://urldefense.com/v3/__https://github.com/Nesvilab/FragPipe/issues/2522*issuecomment-3473411095__;Iw!!HLrAl2XzZ3iCLg!EGlZjWMW92-sL3_BIMQSt-5srcUHnUp_ZialK9tiKb73AJrMAt_qop7lY9hJHIjWtevVtS-rUkrfa0a7TcyfvIAbf5fWO7Y$> Hi Julian, Can you attach the log files from those searches? I don't know any reason Excedion data would not work, but it's always possible that the data format changes with a new instrument in a way we need to explicitly support, but we're happy to do so if that's the case. Best, Dan — Reply to this email directly, view it on GitHub<https://urldefense.com/v3/__https://github.com/Nesvilab/FragPipe/issues/2522*issuecomment-3473411095__;Iw!!HLrAl2XzZ3iCLg!EGlZjWMW92-sL3_BIMQSt-5srcUHnUp_ZialK9tiKb73AJrMAt_qop7lY9hJHIjWtevVtS-rUkrfa0a7TcyfvIAbf5fWO7Y$>, or unsubscribe<https://urldefense.com/v3/__https://github.com/notifications/unsubscribe-auth/BQBE3DFLHYTFLMF52P26DLT32NYZ5AVCNFSM6AAAAACKSG24SOVHI2DSMVQWIX3LMV43OSLTON2WKQ3PNVWWK3TUHMZTINZTGQYTCMBZGU__;!!HLrAl2XzZ3iCLg!EGlZjWMW92-sL3_BIMQSt-5srcUHnUp_ZialK9tiKb73AJrMAt_qop7lY9hJHIjWtevVtS-rUkrfa0a7TcyfvIAbPyFmTQY$>. You are receiving this because you were mentioned.Message ID: ***@***.***> System OS: Windows 11, Architecture: AMD64 Java Info: 17.0.10, OpenJDK 64-Bit Server VM, Eclipse Adoptium .NET Core Info: 6.0.32 Version info: FragPipePlus version 23.2-build3 DIA-Umpire version 2.3.3 diaTracer version 1.3.6 MSFragger version 4.3 Crystal-C version 1.5.9 MSBooster version 1.3.21 Percolator version 3.7.1 PTMProphet version 7.3.0 Metaproteomics version 1.0.1 Philosopher version 5.1.2 PTM-Shepherd version 3.0.2 IonQuant version 1.11.11 TMT-Integrator version 6.1.1 EasyPQP version 0.1.52 DIA-NN version 1.8.2 beta 8 Skyline version N/A Pandas version 2.2.3 Numpy version 1.26.4 LCMS files: Experiment/Group: (if "spectral library generation" is enabled, all files will be analyzed together) - C:\Users\Thermo\Downloads\250821_IO25_DDA_HCD-pd-EThcD_OGlcNc_MsBrain_01.raw DDA 15 commands to execute: CheckCentroid C:\FragPipePlus\FragPipePlus-23.2-build3\jre\bin\java.exe -Xmx110G -cp C:\FragPipePlus\FragPipePlus-23.2-build3\lib\fragpipe-23.2-build3.jar;C:\FragPipePlus\FragPipePlus-23.2-build3\tools\batmass-io-1.35.5.jar org.nesvilab.fragpipe.util.CheckCentroid C:\Users\Thermo\Downloads\250821_IO25_DDA_HCD-pd-EThcD_OGlcNc_MsBrain_01.raw 47 WorkspaceCleanInit [Work dir: C:\Users\Thermo\Downloads\Julian3hybrid] C:\FragPipePlus\FragPipePlus-23.2-build3\tools\Philosopher\philosopher-v5.1.2.exe workspace --clean --nocheck WorkspaceCleanInit [Work dir: C:\Users\Thermo\Downloads\Julian3hybrid] C:\FragPipePlus\FragPipePlus-23.2-build3\tools\Philosopher\philosopher-v5.1.2.exe workspace --init --nocheck --temp C:\Users\Thermo\AppData\Local\Temp\c15cb88e-f8da-44a0-bd3e-c37f27c704d5 MSFragger [Work dir: C:\Users\Thermo\Downloads\Julian3hybrid] C:\FragPipePlus\FragPipePlus-23.2-build3\jre\bin\java.exe -jar -Dfile.encoding=UTF-8 -Xmx110G C:\FragPipePlus\FragPipePlus-23.2-build3\tools\MSFragger-4.3\MSFragger-Commercial-4.3.jar C:\Users\Thermo\Downloads\Julian3hybrid\fragger.params --license C:\FragPipePlus\FragPipePlus-23.2-build3\license.dat C:\Users\Thermo\Downloads\250821_IO25_DDA_HCD-pd-EThcD_OGlcNc_MsBrain_01.raw MSFragger move pepxml C:\FragPipePlus\FragPipePlus-23.2-build3\jre\bin\java.exe -cp C:\FragPipePlus\FragPipePlus-23.2-build3\lib\fragpipe-23.2-build3.jar;/C:/FragPipePlus/FragPipePlus-23.2-build3/lib/batmass-io-1.35.5.jar org.nesvilab.utils.FileMove --no-err C:\Users\Thermo\Downloads\250821_IO25_DDA_HCD-pd-EThcD_OGlcNc_MsBrain_01.pepXML C:\Users\Thermo\Downloads\Julian3hybrid\250821_IO25_DDA_HCD-pd-EThcD_OGlcNc_MsBrain_01.pepXML MSFragger move tsv C:\FragPipePlus\FragPipePlus-23.2-build3\jre\bin\java.exe -cp C:\FragPipePlus\FragPipePlus-23.2-build3\lib\fragpipe-23.2-build3.jar;/C:/FragPipePlus/FragPipePlus-23.2-build3/lib/batmass-io-1.35.5.jar org.nesvilab.utils.FileMove --no-err C:\Users\Thermo\Downloads\250821_IO25_DDA_HCD-pd-EThcD_OGlcNc_MsBrain_01.tsv C:\Users\Thermo\Downloads\Julian3hybrid\250821_IO25_DDA_HCD-pd-EThcD_OGlcNc_MsBrain_01.tsv PeptideProphet [Work dir: C:\Users\Thermo\Downloads\Julian3hybrid] C:\FragPipePlus\FragPipePlus-23.2-build3\tools\Philosopher\philosopher-v5.1.2.exe peptideprophet --nonparam --expectscore --decoyprobs --masswidth 4000.0 --decoy rev_ --database C:\Users\Thermo\Downloads\2025-10-30-decoys-contam-uniprotkb_taxonomy_id_10088_AND_reviewe_2024_11_28.fasta.fas --combine 250821_IO25_DDA_HCD-pd-EThcD_OGlcNc_MsBrain_01.pepXML Rewrite pepxml [Work dir: C:\Users\Thermo\Downloads\Julian3hybrid] C:\FragPipePlus\FragPipePlus-23.2-build3\jre\bin\java.exe -cp C:\FragPipePlus\FragPipePlus-23.2-build3\lib/* org.nesvilab.fragpipe.util.RewritePepxml C:\Users\Thermo\Downloads\Julian3hybrid\interact.pep.xml C:\Users\Thermo\Downloads\250821_IO25_DDA_HCD-pd-EThcD_OGlcNc_MsBrain_01_calibrated.mzML ProteinProphet [Work dir: C:\Users\Thermo\Downloads\Julian3hybrid] C:\FragPipePlus\FragPipePlus-23.2-build3\tools\Philosopher\philosopher-v5.1.2.exe proteinprophet --maxppmdiff 2000000 --output combined C:\Users\Thermo\Downloads\Julian3hybrid\filelist_proteinprophet.txt PhilosopherDbAnnotate [Work dir: C:\Users\Thermo\Downloads\Julian3hybrid] C:\FragPipePlus\FragPipePlus-23.2-build3\tools\Philosopher\philosopher-v5.1.2.exe database --annotate C:\Users\Thermo\Downloads\2025-10-30-decoys-contam-uniprotkb_taxonomy_id_10088_AND_reviewe_2024_11_28.fasta.fas --prefix rev_ PhilosopherFilter [Work dir: C:\Users\Thermo\Downloads\Julian3hybrid] C:\FragPipePlus\FragPipePlus-23.2-build3\tools\Philosopher\philosopher-v5.1.2.exe filter --sequential --prot 0.01 --tag rev_ --pepxml C:\Users\Thermo\Downloads\Julian3hybrid --protxml C:\Users\Thermo\Downloads\Julian3hybrid\combined.prot.xml --razor PhilosopherReport [Work dir: C:\Users\Thermo\Downloads\Julian3hybrid] C:\FragPipePlus\FragPipePlus-23.2-build3\tools\Philosopher\philosopher-v5.1.2.exe report PairScans C:\FragPipePlus\FragPipePlus-23.2-build3\jre\bin\java.exe -Xmx110G -Dlibs.bruker.dir=C:\FragPipePlus\FragPipePlus-23.2-build3\tools\MSFragger-4.3\ext\bruker -Dlibs.thermo.dir=C:\FragPipePlus\FragPipePlus-23.2-build3\tools\MSFragger-4.3\ext\thermo -cp C:\FragPipePlus\FragPipePlus-23.2-build3\lib\fragpipe-23.2-build3.jar;C:\FragPipePlus\FragPipePlus-23.2-build3\tools\batmass-io-1.35.5.jar org.nesvilab.fragpipe.util.PairScans C:\Users\Thermo\Downloads\250821_IO25_DDA_HCD-pd-EThcD_OGlcNc_MsBrain_01.raw 47 ETD ETD false true WorkspaceClean [Work dir: C:\Users\Thermo\Downloads\Julian3hybrid] C:\FragPipePlus\FragPipePlus-23.2-build3\tools\Philosopher\philosopher-v5.1.2.exe workspace --clean --nocheck OPair [Work dir: C:\Users\Thermo\Downloads\Julian3hybrid] C:\FragPipePlus\FragPipePlus-23.2-build3\tools\opair\CMD.exe -b 20.0 -c 20.0 -g HexNAc(1),HexNAc(1)Hex(1),HexNAc(1)NeuAc(1),HexNAc(2)Hex(1),HexNAc(1)Hex(1)NeuAc(1),HexNAc(2)Hex(2),HexNAc(2)Hex(1)NeuAc(1),HexNAc(1)Hex(1)NeuAc(2),HexNAc(2)Hex(2)NeuAc(1),HexNAc(2)Hex(2)Fuc(1)NeuAc(1),HexNAc(2)Hex(2)NeuAc(2),HexNAc(2)Hex(2)Fuc(1)NeuAc(2) -x C:\FragPipePlus\FragPipePlus-23.2-build3\lib\..\tools\Glycan_Databases\glycan_residues.txt -y C:\FragPipePlus\FragPipePlus-23.2-build3\lib\..\tools\Glycan_Databases\glycan_mods.txt -n 4 -z -t 47 -i 0 -j 2 -r C:\Users\Thermo\Downloads\Julian3hybrid\filelist_opair.txt -s C:\Users\Thermo\Downloads\Julian3hybrid\psm.tsv -o C:\Users\Thermo\Downloads\Julian3hybrid ~~~~~~~~~~~~~~~~~~~~~~ Execution order: Cmd: [START], Work dir: [C:\Users\Thermo\Downloads\Julian3hybrid] Cmd: [CheckCentroid], Work dir: [C:\Users\Thermo\Downloads\Julian3hybrid] Cmd: [WorkspaceCleanInit], Work dir: [C:\Users\Thermo\Downloads\Julian3hybrid] Cmd: [MSFragger], Work dir: [C:\Users\Thermo\Downloads\Julian3hybrid] Cmd: [PeptideProphet], Work dir: [C:\Users\Thermo\Downloads\Julian3hybrid] Cmd: [ProteinProphet], Work dir: [C:\Users\Thermo\Downloads\Julian3hybrid] Cmd: [PhilosopherDbAnnotate], Work dir: [C:\Users\Thermo\Downloads\Julian3hybrid] Cmd: [PhilosopherFilter], Work dir: [C:\Users\Thermo\Downloads\Julian3hybrid] Cmd: [PhilosopherReport], Work dir: [C:\Users\Thermo\Downloads\Julian3hybrid] Cmd: [PairScans], Work dir: [C:\Users\Thermo\Downloads\Julian3hybrid] Cmd: [WorkspaceClean], Work dir: [C:\Users\Thermo\Downloads\Julian3hybrid] Cmd: [OPair], Work dir: [C:\Users\Thermo\Downloads\Julian3hybrid] ~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~Sample of C:\Users\Thermo\Downloads\2025-10-30-decoys-contam-uniprotkb_taxonomy_id_10088_AND_reviewe_2024_11_28.fasta.fas~~~~~~~

rev_sp|A0A087WPF7|AUTS2_MOUSE Autism susceptibility gene 2 protein homolog OS=Mus musculus OX=10090 GN=Auts2 PE=1 SV=2 rev_sp|O88703|HCN2_MOUSE Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 2 OS=Mus musculus OX=10090 GN=Hcn2 PE=1 SV=1 rev_sp|P41159|LEP_HUMAN Leptin OS=Homo sapiens OX=9606 GN=LEP PE=1 SV=1 rev_sp|Q07133|H1T_MOUSE Histone H1t OS=Mus musculus OX=10090 GN=H1-6 PE=1 SV=4 rev_sp|Q60770|STXB3_MOUSE Syntaxin-binding protein 3 OS=Mus musculus OX=10090 GN=Stxbp3 PE=1 SV=1 rev_sp|Q7TMM9|TBB2A_MOUSE Tubulin beta-2A chain OS=Mus musculus OX=10090 GN=Tubb2a PE=1 SV=1 rev_sp|Q8BX94|OSBL2_MOUSE Oxysterol-binding protein-related protein 2 OS=Mus musculus OX=10090 GN=Osbpl2 PE=1 SV=1 rev_sp|Q8R4E6|PURG_MOUSE Purine-rich element-binding protein gamma OS=Mus musculus OX=10090 GN=Purg PE=1 SV=1 rev_sp|Q9CQ21|MCTS2_MOUSE Malignant T-cell-amplified sequence 2 OS=Mus musculus OX=10090 GN=Mcts2 PE=1 SV=1 rev_sp|Q9EPW9|TLR6_MOUSE Toll-like receptor 6 OS=Mus musculus OX=10090 GN=Tlr6 PE=1 SV=2 rev_sp|S4R2K0|DEFM_MOUSE Peptide deformylase, mitochondrial OS=Mus musculus OX=10090 GN=Pdf PE=3 SV=1 sp|O88676|MMP23_MOUSE Matrix metalloproteinase-23 OS=Mus musculus OX=10090 GN=Mmp23 PE=2 SV=1 sp|P41105|RL28_MOUSE Large ribosomal subunit protein eL28 OS=Mus musculus OX=10090 GN=Rpl28 PE=1 SV=2 sp|Q06985|SIA1B_MOUSE E3 ubiquitin-protein ligase SIAH1B OS=Mus musculus OX=10090 GN=Siah1b PE=2 SV=2 sp|Q60759|GCDH_MOUSE Glutaryl-CoA dehydrogenase, mitochondrial OS=Mus musculus OX=10090 GN=Gcdh PE=1 SV=2 sp|Q7TMF5|SPA12_MOUSE Serpin A12 OS=Mus musculus OX=10090 GN=Serpina12 PE=1 SV=2 sp|Q8BX51|SGCZ_MOUSE Zeta-sarcoglycan OS=Mus musculus OX=10090 GN=Sgcz PE=2 SV=1 sp|Q8R493|ACHB4_MOUSE Neuronal acetylcholine receptor subunit beta-4 OS=Mus musculus OX=10090 GN=Chrnb4 PE=2 SV=1 sp|Q9CQ06|RM24_MOUSE Large ribosomal subunit protein uL24m OS=Mus musculus OX=10090 GN=Mrpl24 PE=1 SV=1 sp|Q9EPU4|CPSF1_MOUSE Cleavage and polyadenylation specificity factor subunit 1 OS=Mus musculus OX=10090 GN=Cpsf1 PE=1 SV=1 sp|Q9Z320|K1C27_MOUSE Keratin, type I cytoskeletal 27 OS=Mus musculus OX=10090 GN=Krt27 PE=1 SV=1

~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~ fragpipe.config ~~~~~~~~~ # FragPipePlus v23.2-build3ui state cache # Please edit the following path to point to the correct location. # In Windows, please replace single '\' with '\\' database.db-path=C\:\\Users\\Thermo\\Downloads\\2025-10-30-decoys-contam-uniprotkb_taxonomy_id_10088_AND_reviewe_2024_11_28.fasta.fas crystalc.run-crystalc=false database.decoy-tag=rev_ diann.channel-normalization-strategy=0 diann.cmd-opts= diann.fragpipe.cmd-opts= diann.generate-msstats=true diann.heavy= diann.library= diann.light= diann.medium= diann.min-site-prob=0.75 diann.mod-tag= diann.q-value=0.01 diann.quantification-strategy=3 diann.quantification-strategy-2=2 diann.run-dia-nn=false diann.run-dia-plex=false diann.run-specific-protein-q-value=false diann.unrelated-runs=false diann.use-predicted-spectra=false diatracer.corr-threshold=0.3 diatracer.delta-apex-im=0.01 diatracer.delta-apex-rt=3 diatracer.mass-defect-filter=true diatracer.mass-defect-offset=0.1 diatracer.rf-max=500 diatracer.run-diatracer=false diatracer.write-intermediate-files=false diaumpire.AdjustFragIntensity=true diaumpire.BoostComplementaryIon=false diaumpire.CorrThreshold=0 diaumpire.DeltaApex=0.2 diaumpire.ExportPrecursorPeak=false diaumpire.Q1=true diaumpire.Q2=true diaumpire.Q3=true diaumpire.RFmax=500 diaumpire.RPmax=25 diaumpire.RTOverlap=0.3 diaumpire.SE.EstimateBG=false diaumpire.SE.IsoPattern=0.3 diaumpire.SE.MS1PPM=10 diaumpire.SE.MS2PPM=20 diaumpire.SE.MS2SN=1.1 diaumpire.SE.MassDefectFilter=true diaumpire.SE.MassDefectOffset=0.1 diaumpire.SE.NoMissedScan=1 diaumpire.SE.SN=1.1 diaumpire.run-diaumpire=false fpop.coadaptr.fpop.fpop_masses= fpop.coadaptr.fpop.run-fpop-coadaptr=false fpop.fpop-tmt=false fpop.fragpipe.fpop.fpop-tmt=false fpop.fragpipe.fpop.label_control= fpop.fragpipe.fpop.label_fpop= fpop.fragpipe.fpop.region_size=1 fpop.fragpipe.fpop.run-fpop=false fpop.fragpipe.fpop.subtract-control=false fpop.label_control= fpop.label_fpop= fpop.region_size=1 fpop.run-fpop=false fpop.subtract-control=false fragpipe-config.bin-diann=C\:\\FragPipePlus\\FragPipePlus-23.2-build3\\tools\\diann\\1.8.2_beta_8\\windows\\DiaNN.exe fragpipe-config.bin-python=C\:\\FragPipePlus\\FragPipePlus-23.2-build3\\python\\python.exe fragpipe-config.tools-folder=C\:\\FragPipePlus\\FragPipePlus-23.2-build3\\tools freequant.mz-tol=10 freequant.rt-tol=0.4 freequant.run-freequant=false ionquant.excludemods= ionquant.formula= ionquant.heavy= ionquant.imtol=0.05 ionquant.intensitymode=0 ionquant.ionfdr=0.01 ionquant.light= ionquant.locprob=0.75 ionquant.maxlfq=1 ionquant.mbr=0 ionquant.mbrimtol=0.05 ionquant.mbrmincorr=0 ionquant.mbrrttol=1 ionquant.mbrtoprun=10 ionquant.medium= ionquant.minfreq=0 ionquant.minions=1 ionquant.minisotopes=2 ionquant.minscans=3 ionquant.mztol=10 ionquant.normalization=1 ionquant.peptidefdr=1 ionquant.proteinfdr=1 ionquant.requantify=1 ionquant.rttol=0.4 ionquant.run-ionquant=true ionquant.tp=0 ionquant.uniqueness=0 ionquant.use-labeling=false ionquant.use-lfq=true ionquant.writeindex=0 metaproteomics.cmd-line-opts= metaproteomics.delta-hyperscore=0.0 metaproteomics.host-name=Homo sapiens metaproteomics.iterations=3 metaproteomics.min-pept-cnt-per-prot=1 metaproteomics.min-uniq-pept-cnt=3 metaproteomics.min-uniq-pept-cnt-per-prot=1 metaproteomics.qvalue=0.01 metaproteomics.run-metaproteomics=false msbooster.find-best-im-model=false msbooster.find-best-rt-model=false msbooster.find-best-spectra-model=false msbooster.fragmentation-type=0 msbooster.im-model=DIA-NN msbooster.koina-url= msbooster.predict-im=true msbooster.predict-rt=true msbooster.predict-spectra=true msbooster.rt-model=DIA-NN msbooster.run-msbooster=false msbooster.spectra-model=DIA-NN msbooster.spectral-library-path= msfragger.Y_type_masses=0 203.07937 365.1322 406.15874 568.21156 730.26438 892.3172 349.137279 msfragger.activation_types=all msfragger.allowed_missed_cleavage_1=2 msfragger.allowed_missed_cleavage_2=2 msfragger.analyzer_types=all msfragger.calibrate_mass=2 msfragger.check_spectral_files=true msfragger.clip_nTerm_M=true msfragger.deisotope=1 msfragger.delta_mass_exclude_ranges=(-1.5,3.5) msfragger.deneutralloss=1 msfragger.diagnostic_fragments=204.086646 186.076086 168.065526 366.139466 144.0656 138.055 512.197375 292.1026925 274.0921325 657.2349 243.026426 405.079246 485.045576 308.09761 msfragger.diagnostic_intensity_filter=0.03 msfragger.digest_max_length=50 msfragger.digest_min_length=7 msfragger.fragment_ion_series=b,y,c,z,Y msfragger.fragment_mass_tolerance=20 msfragger.fragment_mass_units=1 msfragger.group_variable=0 msfragger.intensity_transform=0 msfragger.ion_series_definitions= msfragger.isotope_error=0/1/2 msfragger.labile_search_mode=labile msfragger.localize_delta_mass=true msfragger.mass_diff_to_variable_mod=0 msfragger.mass_offsets=0 203.0793762207 365.1322021484 494.1748046875 568.2115783691 656.2276306152 730.2644042969 859.3070068359 947.323059082 1021.3598327637 1167.4177398682 1312.4552612305 1458.513168335 406.1587524414 697.2541809082 771.2909545898 933.3437805176 1062.3863830566 1150.4024353027 1224.4392089844 1370.4971160889 1515.5346374512 1661.5925445557 1095.3966064453 1386.4920349121 1532.5499420166 1677.5874633789 1823.6453704834 988.349609375 1353.4818115234 1441.4978637695 1806.630065918 1952.6879730225 1136.4231567383 1298.475982666 1427.5185852051 1589.5714111328 1735.6293182373 1880.6668395996 2026.7247467041 1603.5506896973 1968.6828918457 2114.7407989502 1460.5288085938 1751.6242370605 1897.682144165 2042.7196655273 2188.7775726318 1718.6140136719 2171.7622680664 2317.8201751709 1894.6461181641 2259.7783203125 2405.836227417 2333.8150939941 2479.8730010986 2334.8354797363 2625.9309082031 2624.9105224609 2770.9684295654 2917.0263366699 609.2381286621 900.3335571289 974.3703308105 1265.4657592773 1573.5764923096 1864.6719207764 1191.4289855957 1556.5611877441 1644.5772399902 2009.7094421387 2155.7673492432 1339.502532959 1501.5553588867 1630.5979614258 1792.6507873535 1938.708694458 2083.7462158203 2229.8041229248 1663.6081848145 1954.7036132812 2100.7615203857 2245.799041748 2391.8569488525 1921.6933898926 2374.8416442871 2520.8995513916 2097.7254943848 2462.8576965332 2608.9156036377 2536.8944702148 2682.9523773193 2537.914855957 2829.0102844238 2827.9898986816 2974.0478057861 1825.6610107422 2116.756439209 2262.8143463135 2407.8518676758 2553.9097747803 2698.9472961426 2845.0052032471 2699.9676818848 2991.0631103516 2990.0427246094 1482.5244140625 1847.6566162109 1935.672668457 2300.8048706055 2446.86277771 2212.7888183594 2665.9370727539 2811.9949798584 2388.8209228516 2753.953125 2900.0110321045 1704.6347351074 1866.6875610352 1995.7301635742 2157.782989502 2303.8408966064 2448.8784179688 2594.9363250732 2028.7403869629 2319.8358154297 2465.8937225342 2610.9312438965 2756.989151001 2286.825592041 2739.9738464355 2886.03175354 2902.0266723633 2903.0470581055 2550.8737487793 2916.0059509277 2190.7932128906 2481.8886413574 2627.9465484619 2772.9840698242 2919.0419769287 2577.9210205078 2841.9691772461 812.3175048828 1103.4129333496 1177.4497070312 1468.545135498 1776.6558685303 2067.7512969971 1394.5083618164 1759.6405639648 2358.8467254639 1542.5819091797 1833.6773376465 2141.7880706787 2432.8834991455 2124.7727661133 2723.9789276123 2740.9942321777 1685.6037902832 2050.7359924316 2138.7520446777 2503.8842468262 2649.9421539307 2415.8681945801 2869.0164489746 2591.9002990723 2957.0325012207 1907.7141113281 2069.7669372559 2198.8095397949 2360.8623657227 2506.9202728271 2651.9577941895 2798.0157012939 2231.8197631836 2522.9151916504 2668.9730987549 2814.0106201172 2960.0685272217 2489.9049682617 2943.0532226562 2393.8725891113 2684.9680175781 2831.0259246826 2976.0634460449 2781.0003967285 2555.9254150391 2847.0208435059 2993.0787506104 1976.69921875 2341.8314208984 2429.8474731445 2794.979675293 2941.0375823975 2706.9636230469 2882.9957275391 2272.8463134766 2434.8991394043 2563.9417419434 2725.9945678711 2872.0524749756 2596.951965332 2888.0473937988 2855.0371704102 2759.0047912598 2921.0576171875 msfragger.mass_offsets_detailed= msfragger.max_fragment_charge=2 msfragger.max_variable_mods_combinations=5000 msfragger.max_variable_mods_per_peptide=3 msfragger.min_fragments_modelling=2 msfragger.min_matched_fragments=4 msfragger.min_sequence_matches=2 msfragger.minimum_peaks=15 msfragger.minimum_ratio=0.00 msfragger.misc.fragger.clear-mz-hi=0 msfragger.misc.fragger.clear-mz-lo=0 msfragger.misc.fragger.digest-mass-hi=5000 msfragger.misc.fragger.digest-mass-lo=400 msfragger.misc.fragger.enzyme-dropdown-1=stricttrypsin msfragger.misc.fragger.enzyme-dropdown-2=null msfragger.misc.fragger.precursor-charge-hi=4 msfragger.misc.fragger.precursor-charge-lo=1 msfragger.misc.fragger.remove-precursor-range-hi=1.5 msfragger.misc.fragger.remove-precursor-range-lo=-1.5 msfragger.misc.slice-db=1 msfragger.num_enzyme_termini=2 msfragger.output_format=tsv_pepXML msfragger.output_max_expect=50 msfragger.output_report_topN=1 msfragger.output_report_topN_dda_plus=5 msfragger.output_report_topN_dia1=5 msfragger.override_charge=false msfragger.precursor_mass_lower=-20 msfragger.precursor_mass_mode=corrected msfragger.precursor_mass_units=1 msfragger.precursor_mass_upper=20 msfragger.precursor_true_tolerance=20 msfragger.precursor_true_units=1 msfragger.remainder_fragment_masses= msfragger.remove_precursor_peak=2 msfragger.report_alternative_proteins=true msfragger.require_precursor=true msfragger.restrict_deltamass_to=ST- msfragger.reuse_dia_fragment_peaks=false msfragger.run-msfragger=true msfragger.search_enzyme_cut_1=KR msfragger.search_enzyme_cut_2= msfragger.search_enzyme_name_1=stricttrypsin msfragger.search_enzyme_name_2=null msfragger.search_enzyme_nocut_1= msfragger.search_enzyme_nocut_2= msfragger.search_enzyme_sense_1=C msfragger.search_enzyme_sense_2=C msfragger.table.fix-mods=0.0,C-Term Peptide,true,-1; 0.0,N-Term Peptide,true,-1; 0.0,C-Term Protein,true,-1; 0.0,N-Term Protein,true,-1; 0.0,G (glycine),true,-1; 0.0,A (alanine),true,-1; 0.0,S (serine),true,-1; 0.0,P (proline),true,-1; 0.0,V (valine),true,-1; 0.0,T (threonine),true,-1; 57.02146,C (cysteine),true,-1; 0.0,L (leucine),true,-1; 0.0,I (isoleucine),true,-1; 0.0,N (asparagine),true,-1; 0.0,D (aspartic acid),true,-1; 0.0,Q (glutamine),true,-1; 0.0,K (lysine),true,-1; 0.0,E (glutamic acid),true,-1; 0.0,M (methionine),true,-1; 0.0,H (histidine),true,-1; 0.0,F (phenylalanine),true,-1; 0.0,R (arginine),true,-1; 0.0,Y (tyrosine),true,-1; 0.0,W (tryptophan),true,-1; 0.0,B ,true,-1; 0.0,J,true,-1; 0.0,O,true,-1; 0.0,U,true,-1; 0.0,X,true,-1; 0.0,Z,true,-1 msfragger.table.var-mods=15.9949,M,true,3; 42.0106,[^,true,1; 79.96633,STY,false,3; -17.0265,nQnC,false,1; -18.0106,nE,false,1; 4.025107,K,false,3; 6.020129,R,false,3 msfragger.track_zero_topN=0 msfragger.use_all_mods_in_first_search=false msfragger.use_detailed_offsets=false msfragger.use_topN_peaks=300 msfragger.write_calibrated_mzml=true msfragger.write_uncalibrated_mgf=false msfragger.zero_bin_accept_expect=0 msfragger.zero_bin_mult_expect=1 opair.activation1=ETD opair.activation2=ETD opair.allowed_sites= opair.filterOxonium=false opair.glyco_db=HexNAc(1),HexNAc(1)Hex(1),HexNAc(1)NeuAc(1),HexNAc(2)Hex(1),HexNAc(1)Hex(1)NeuAc(1),HexNAc(2)Hex(2),HexNAc(2)Hex(1)NeuAc(1),HexNAc(1)Hex(1)NeuAc(2),HexNAc(2)Hex(2)NeuAc(1),HexNAc(2)Hex(2)Fuc(1)NeuAc(1),HexNAc(2)Hex(2)NeuAc(2),HexNAc(2)Hex(2)Fuc(1)NeuAc(2) opair.max_glycans=4 opair.max_isotope_error=2 opair.min_isotope_error=0 opair.ms1_tol=20 opair.ms2_tol=20 opair.oxonium_filtering_file= opair.oxonium_minimum_intensity=0.01 opair.reverse_scan_order=false opair.run-opair=true opair.single_scan_type=true peptide-prophet.cmd-opts=--nonparam --expectscore --decoyprobs --masswidth 4000.0 peptide-prophet.combine-pepxml=true peptide-prophet.run-peptide-prophet=true percolator.cmd-opts=--only-psms --no-terminate --post-processing-tdc percolator.keep-tsv-files=false percolator.min-prob=0.5 percolator.run-percolator=false phi-report.dont-use-prot-proph-file=false phi-report.filter=--sequential --prot 0.01 phi-report.pep-level-summary=false phi-report.print-decoys=false phi-report.prot-level-summary=false phi-report.remove-contaminants=false phi-report.run-report=true protein-prophet.cmd-opts=--maxppmdiff 2000000 protein-prophet.run-protein-prophet=true ptmprophet.cmdline= ptmprophet.override-defaults=false ptmprophet.run-ptmprophet=false ptmshepherd.adv_params=false ptmshepherd.annotate_assigned_mods=false ptmshepherd.annotation-common=false ptmshepherd.annotation-custom=false ptmshepherd.annotation-glyco=true ptmshepherd.annotation-unimod=false ptmshepherd.annotation_file= ptmshepherd.annotation_tol=0.01 ptmshepherd.cap_y_ions=0,203.07937,406.15874,568.21156,730.26438,349.137279 ptmshepherd.decoy_type=1 ptmshepherd.diag_ions=204.086646,186.076086,168.065526,366.139466,144.0656,138.055,512.197375,292.1026925,274.0921325,657.2349,243.026426,405.079246,485.045576,308.09761 ptmshepherd.diagmine_diagMinFoldChange=3.0 ptmshepherd.diagmine_diagMinSpecDiff=25 ptmshepherd.diagmine_fragMinFoldChange=3.0 ptmshepherd.diagmine_fragMinPropensity=12.5 ptmshepherd.diagmine_fragMinSpecDiff=25 ptmshepherd.diagmine_minIonsPerSpec=2 ptmshepherd.diagmine_minPeps=25 ptmshepherd.diagmine_pepMinFoldChange=3.0 ptmshepherd.diagmine_pepMinSpecDiff=25 ptmshepherd.glyco_fdr=0.01 ptmshepherd.glyco_isotope_max=3 ptmshepherd.glyco_isotope_min=-1 ptmshepherd.glyco_ppm_tol=50 ptmshepherd.glycodatabase=HexNAc(1),HexNAc(1)Hex(1),HexNAc(1)NeuAc(1),HexNAc(2)Hex(1),HexNAc(1)Hex(1)NeuAc(1),HexNAc(2)Hex(2),HexNAc(2)Hex(1)NeuAc(1),HexNAc(1)Hex(1)NeuAc(2),HexNAc(2)Hex(2)NeuAc(1),HexNAc(2)Hex(2)Fuc(1)NeuAc(1),HexNAc(2)Hex(2)NeuAc(2),HexNAc(2)Hex(2)Fuc(1)NeuAc(2),HexNAc(2),HexNAc(2)NeuAc(1),HexNAc(3)Hex(1),HexNAc(3)Hex(2),HexNAc(3)Hex(1)NeuAc(1),HexNAc(2)Hex(1)NeuAc(2),HexNAc(3)Hex(2)NeuAc(1),HexNAc(3)Hex(2)Fuc(1)NeuAc(1),HexNAc(3)Hex(2)NeuAc(2),HexNAc(3)Hex(2)Fuc(1)NeuAc(2),HexNAc(3)Hex(3),HexNAc(3)Hex(3)NeuAc(1),HexNAc(3)Hex(3)Fuc(1)NeuAc(1),HexNAc(3)Hex(3)NeuAc(2),HexNAc(3)Hex(3)Fuc(1)NeuAc(2),HexNAc(2)NeuAc(2),HexNAc(3)Hex(1)NeuAc(2),HexNAc(2)Hex(1)NeuAc(3),HexNAc(3)Hex(2)NeuAc(3),HexNAc(3)Hex(2)Fuc(1)NeuAc(3),HexNAc(4)Hex(2),HexNAc(4)Hex(3),HexNAc(4)Hex(2)NeuAc(1),HexNAc(4)Hex(3)NeuAc(1),HexNAc(4)Hex(3)Fuc(1)NeuAc(1),HexNAc(4)Hex(3)NeuAc(2),HexNAc(4)Hex(3)Fuc(1)NeuAc(2),HexNAc(2)Hex(2)NeuAc(3),HexNAc(3)Hex(3)NeuAc(3),HexNAc(3)Hex(3)Fuc(1)NeuAc(3),HexNAc(4)Hex(4),HexNAc(4)Hex(4)NeuAc(1),HexNAc(4)Hex(4)Fuc(1)NeuAc(1),HexNAc(4)Hex(4)NeuAc(2),HexNAc(4)Hex(4)Fuc(1)NeuAc(2),HexNAc(4)Hex(2)NeuAc(2),HexNAc(4)Hex(3)NeuAc(3),HexNAc(4)Hex(3)Fuc(1)NeuAc(3),HexNAc(2)Hex(2)NeuAc(4),HexNAc(3)Hex(3)NeuAc(4),HexNAc(3)Hex(3)Fuc(1)NeuAc(4),HexNAc(4)Hex(4)NeuAc(3),HexNAc(4)Hex(4)Fuc(1)NeuAc(3),HexNAc(4)Hex(4)Fuc(2)NeuAc(2),HexNAc(4)Hex(4)Fuc(2)NeuAc(3),HexNAc(4)Hex(4)NeuAc(4),HexNAc(4)Hex(4)Fuc(1)NeuAc(4),HexNAc(4)Hex(4)Fuc(2)NeuAc(4),HexNAc(3),HexNAc(3)NeuAc(1),HexNAc(4)Hex(1),HexNAc(4)Hex(1)NeuAc(1),HexNAc(4)Hex(2)Fuc(1)NeuAc(1),HexNAc(4)Hex(2)Fuc(1)NeuAc(2),HexNAc(3)NeuAc(2),HexNAc(4)Hex(1)NeuAc(2),HexNAc(3)Hex(1)NeuAc(3),HexNAc(4)Hex(2)NeuAc(3),HexNAc(4)Hex(2)Fuc(1)NeuAc(3),HexNAc(5)Hex(2),HexNAc(5)Hex(3),HexNAc(5)Hex(2)NeuAc(1),HexNAc(5)Hex(3)NeuAc(1),HexNAc(5)Hex(3)Fuc(1)NeuAc(1),HexNAc(5)Hex(3)NeuAc(2),HexNAc(5)Hex(3)Fuc(1)NeuAc(2),HexNAc(5)Hex(4),HexNAc(5)Hex(4)NeuAc(1),HexNAc(5)Hex(4)Fuc(1)NeuAc(1),HexNAc(5)Hex(4)NeuAc(2),HexNAc(5)Hex(4)Fuc(1)NeuAc(2),HexNAc(5)Hex(2)NeuAc(2),HexNAc(5)Hex(3)NeuAc(3),HexNAc(5)Hex(3)Fuc(1)NeuAc(3),HexNAc(3)Hex(2)NeuAc(4),HexNAc(4)Hex(3)NeuAc(4),HexNAc(4)Hex(3)Fuc(1)NeuAc(4),HexNAc(5)Hex(4)NeuAc(3),HexNAc(5)Hex(4)Fuc(1)NeuAc(3),HexNAc(5)Hex(4)Fuc(2)NeuAc(2),HexNAc(5)Hex(4)Fuc(2)NeuAc(3),HexNAc(5)Hex(4)NeuAc(4),HexNAc(5)Hex(4)Fuc(1)NeuAc(4),HexNAc(5)Hex(5),HexNAc(5)Hex(5)NeuAc(1),HexNAc(5)Hex(5)Fuc(1)NeuAc(1),HexNAc(5)Hex(5)NeuAc(2),HexNAc(5)Hex(5)Fuc(1)NeuAc(2),HexNAc(5)Hex(5)NeuAc(3),HexNAc(5)Hex(5)Fuc(1)NeuAc(3),HexNAc(5)Hex(5)Fuc(2)NeuAc(2),HexNAc(5)Hex(5)Fuc(2)NeuAc(3),HexNAc(5)Hex(5)NeuAc(4),HexNAc(3)NeuAc(3),HexNAc(4)Hex(1)NeuAc(3),HexNAc(3)Hex(1)NeuAc(4),HexNAc(4)Hex(2)NeuAc(4),HexNAc(4)Hex(2)Fuc(1)NeuAc(4),HexNAc(5)Hex(2)NeuAc(3),HexNAc(5)Hex(3)NeuAc(4),HexNAc(5)Hex(3)Fuc(1)NeuAc(4),HexNAc(3)Hex(2)NeuAc(5),HexNAc(4)Hex(3)NeuAc(5),HexNAc(4)Hex(3)Fuc(1)NeuAc(5),HexNAc(6)Hex(3),HexNAc(6)Hex(4),HexNAc(6)Hex(3)NeuAc(1),HexNAc(6)Hex(4)NeuAc(1),HexNAc(6)Hex(4)Fuc(1)NeuAc(1),HexNAc(6)Hex(4)NeuAc(2),HexNAc(6)Hex(4)Fuc(1)NeuAc(2),HexNAc(6)Hex(5),HexNAc(6)Hex(5)NeuAc(1),HexNAc(6)Hex(5)Fuc(1)NeuAc(1),HexNAc(6)Hex(5)NeuAc(2),HexNAc(6)Hex(5)Fuc(1)NeuAc(2),HexNAc(6)Hex(3)NeuAc(2),HexNAc(6)Hex(4)NeuAc(3),HexNAc(6)Hex(4)Fuc(1)NeuAc(3),HexNAc(6)Hex(5)NeuAc(3),HexNAc(6)Hex(5)Fuc(2)NeuAc(2),HexNAc(3)Hex(3)NeuAc(5),HexNAc(4)Hex(4)NeuAc(5),HexNAc(6)Hex(6),HexNAc(6)Hex(6)NeuAc(1),HexNAc(6)Hex(6)Fuc(1)NeuAc(1),HexNAc(6)Hex(6)NeuAc(2),HexNAc(6)Hex(6)Fuc(1)NeuAc(2),HexNAc(6)Hex(3)NeuAc(3),HexNAc(3)Hex(3)NeuAc(6),HexNAc(4),HexNAc(4)NeuAc(1),HexNAc(5)Hex(1),HexNAc(5)Hex(1)NeuAc(1),HexNAc(5)Hex(2)Fuc(1)NeuAc(1),HexNAc(5)Hex(2)Fuc(1)NeuAc(2),HexNAc(4)NeuAc(2),HexNAc(5)Hex(1)NeuAc(2),HexNAc(5)Hex(2)Fuc(1)NeuAc(3),HexNAc(6)Hex(2),HexNAc(6)Hex(2)NeuAc(1),HexNAc(6)Hex(3)Fuc(1)NeuAc(1),HexNAc(6)Hex(3)Fuc(1)NeuAc(2),HexNAc(6)Hex(2)NeuAc(2),HexNAc(6)Hex(3)Fuc(1)NeuAc(3),HexNAc(6)Hex(4)Fuc(2)NeuAc(2),HexNAc(4)NeuAc(3),HexNAc(5)Hex(1)NeuAc(3),HexNAc(4)Hex(1)NeuAc(4),HexNAc(5)Hex(2)NeuAc(4),HexNAc(5)Hex(2)Fuc(1)NeuAc(4),HexNAc(6)Hex(2)NeuAc(3),HexNAc(6)Hex(3)NeuAc(4),HexNAc(4)Hex(2)NeuAc(5),HexNAc(5)Hex(3)NeuAc(5),HexNAc(7)Hex(3),HexNAc(7)Hex(4),HexNAc(7)Hex(3)NeuAc(1),HexNAc(7)Hex(4)NeuAc(1),HexNAc(7)Hex(4)Fuc(1)NeuAc(1),HexNAc(7)Hex(4)NeuAc(2),HexNAc(7)Hex(4)Fuc(1)NeuAc(2),HexNAc(7)Hex(5),HexNAc(7)Hex(5)NeuAc(1),HexNAc(7)Hex(5)Fuc(1)NeuAc(1),HexNAc(7)Hex(5)NeuAc(2),HexNAc(7)Hex(5)Fuc(1)NeuAc(2),HexNAc(7)Hex(3)NeuAc(2),HexNAc(7)Hex(4)NeuAc(3),HexNAc(7)Hex(6),HexNAc(7)Hex(6)NeuAc(1),HexNAc(7)Hex(6)Fuc(1)NeuAc(1),HexNAc(7)Hex(6)NeuAc(2),HexNAc(7)Hex(3)NeuAc(3),HexNAc(7)Hex(7),HexNAc(7)Hex(7)NeuAc(1),HexNAc(7)Hex(7)Fuc(1)NeuAc(1),HexNAc(4)NeuAc(4),HexNAc(5)Hex(1)NeuAc(4),HexNAc(4)Hex(1)NeuAc(5),HexNAc(5)Hex(2)NeuAc(5),HexNAc(5)Hex(2)Fuc(1)NeuAc(5),HexNAc(6)Hex(2)NeuAc(4),HexNAc(4)Hex(2)NeuAc(6),HexNAc(8)Hex(4),HexNAc(8)Hex(5),HexNAc(8)Hex(4)NeuAc(1),HexNAc(8)Hex(5)NeuAc(1),HexNAc(8)Hex(5)Fuc(1)NeuAc(1),HexNAc(8)Hex(6),HexNAc(8)Hex(6)NeuAc(1),HexNAc(8)Hex(4)NeuAc(2),HexNAc(8)Hex(7),HexNAc(8)Hex(8) ptmshepherd.histo_smoothbins=2 ptmshepherd.iontype_a=false ptmshepherd.iontype_b=true ptmshepherd.iontype_c=true ptmshepherd.iontype_x=false ptmshepherd.iontype_y=true ptmshepherd.iontype_z=true ptmshepherd.iterloc_maxEpoch=100 ptmshepherd.iterloc_mode=false ptmshepherd.localization_allowed_res=ST ptmshepherd.n_glyco=false ptmshepherd.normalization-psms=true ptmshepherd.normalization-scans=false ptmshepherd.output_extended=false ptmshepherd.peakpicking_mass_units=0 ptmshepherd.peakpicking_minPsm=10 ptmshepherd.peakpicking_promRatio=0.3 ptmshepherd.peakpicking_width=0.002 ptmshepherd.precursor_mass_units=0 ptmshepherd.precursor_tol=0.01 ptmshepherd.print_decoys=false ptmshepherd.print_full_glyco_params=true ptmshepherd.prob_mass=1.0 ptmshepherd.remainder_masses=203.07937 ptmshepherd.remove_glycan_delta_mass=true ptmshepherd.run-shepherd=false ptmshepherd.run_diagextract_mode=false ptmshepherd.run_diagmine_mode=false ptmshepherd.run_glyco_mode=false ptmshepherd.spectra_condPeaks=150 ptmshepherd.spectra_condRatio=0.0001 ptmshepherd.spectra_maxPrecursorCharge=4 ptmshepherd.spectra_maxfragcharge=2 ptmshepherd.spectra_ppmtol=20 ptmshepherd.use_msfragger_localization=false ptmshepherd.varmod_masses= quantitation.run-label-free-quant=false run-psm-validation=true run-validation-tab=true saintexpress.cmd-opts= saintexpress.fragpipe.cmd-opts= saintexpress.max-replicates=10 saintexpress.run-saint-express=false saintexpress.virtual-controls=100 skyline.run-skyline=false skyline.skyline=true skyline.skyline-custom=false skyline.skyline-custom-path= skyline.skyline-daily=false skyline.skyline-fragment-tolerance=10 skyline.skyline-mode=0 skyline.skyline-mods-mode=Default skyline.skyline-precursor-tolerance=10 skyline.use-ssl=false speclibgen.convert-pepxml=false speclibgen.convert-psm=true speclibgen.easypqp.extras.max_delta_ppm=15 speclibgen.easypqp.extras.max_delta_unimod=0.02 speclibgen.easypqp.extras.max_glycan_qval=1 speclibgen.easypqp.extras.rt_lowess_fraction=0 speclibgen.easypqp.fragment.a=false speclibgen.easypqp.fragment.b=true speclibgen.easypqp.fragment.c=false speclibgen.easypqp.fragment.x=false speclibgen.easypqp.fragment.y=true speclibgen.easypqp.fragment.z=false speclibgen.easypqp.ignore_unannotated=false speclibgen.easypqp.im-cal=Automatic selection of a run as reference IM speclibgen.easypqp.labile_mode=O-glyco speclibgen.easypqp.neutral_loss=false speclibgen.easypqp.rt-cal=noiRT speclibgen.easypqp.select-file.text= speclibgen.easypqp.select-im-file.text= speclibgen.keep-intermediate-files=false speclibgen.run-speclibgen=false tab-run.delete_calibrated_mzml=false tab-run.delete_temp_files=false tab-run.export_matched_fragments=false tab-run.sub_mzml_prob_threshold=0.5 tab-run.write_sub_mzml=false tmtintegrator.add_Ref=-1 tmtintegrator.aggregation_method=0 tmtintegrator.allow_overlabel=true tmtintegrator.allow_unlabeled=true tmtintegrator.best_psm=true tmtintegrator.channel_num=TMT-6 tmtintegrator.extraction_tool=IonQuant tmtintegrator.glyco_qval=0.01 tmtintegrator.groupby=-1 tmtintegrator.log2transformed=true tmtintegrator.max_pep_prob_thres=0 tmtintegrator.min_ntt=0 tmtintegrator.min_pep_prob=0.9 tmtintegrator.min_percent=0.05 tmtintegrator.min_purity=0.5 tmtintegrator.min_resolution=0 tmtintegrator.min_site_prob=-1 tmtintegrator.min_snr=0 tmtintegrator.mod_tag=none tmtintegrator.ms1_int=true tmtintegrator.outlier_removal=true tmtintegrator.philosopher-msstats=false tmtintegrator.print_RefInt=false tmtintegrator.prot_exclude=none tmtintegrator.prot_norm=0 tmtintegrator.psm_norm=false tmtintegrator.quant_level=2 tmtintegrator.ref_d_tag=Pool tmtintegrator.ref_tag=Bridge tmtintegrator.run-tmtintegrator=false tmtintegrator.tolerance=20 tmtintegrator.top3_pep=true tmtintegrator.unique_gene=0 tmtintegrator.unique_pep=false tmtintegrator.use_glycan_composition=true workdir=C\:\\Users\\Thermo\\Downloads\\Julian3hybrid workflow.input.data-type.im-ms=false workflow.input.data-type.regular-ms=true workflow.misc.save-sdrf=true workflow.misc.sdrf-type=Default workflow.ram=0 workflow.threads=47 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ CheckCentroid C:\FragPipePlus\FragPipePlus-23.2-build3\jre\bin\java.exe -Xmx110G -cp C:\FragPipePlus\FragPipePlus-23.2-build3\lib\fragpipe-23.2-build3.jar;C:\FragPipePlus\FragPipePlus-23.2-build3\tools\batmass-io-1.35.5.jar org.nesvilab.fragpipe.util.CheckCentroid C:\Users\Thermo\Downloads\250821_IO25_DDA_HCD-pd-EThcD_OGlcNc_MsBrain_01.raw 47 Done in 0.0 s. Process 'CheckCentroid' finished, exit code: 0 WorkspaceCleanInit [Work dir: C:\Users\Thermo\Downloads\Julian3hybrid] C:\FragPipePlus\FragPipePlus-23.2-build3\tools\Philosopher\philosopher-v5.1.2.exe workspace --clean --nocheck time="07:53:47" level=info msg="Executing Workspace v5.1.2" time="07:53:47" level=info msg="Removing workspace" time="07:53:47" level=info msg=Done Process 'WorkspaceCleanInit' finished, exit code: 0 WorkspaceCleanInit [Work dir: C:\Users\Thermo\Downloads\Julian3hybrid] C:\FragPipePlus\FragPipePlus-23.2-build3\tools\Philosopher\philosopher-v5.1.2.exe workspace --init --nocheck --temp C:\Users\Thermo\AppData\Local\Temp\c15cb88e-f8da-44a0-bd3e-c37f27c704d5 time="07:53:48" level=info msg="Executing Workspace v5.1.2" time="07:53:48" level=info msg="Creating workspace" time="07:53:48" level=info msg=Done Process 'WorkspaceCleanInit' finished, exit code: 0 MSFragger [Work dir: C:\Users\Thermo\Downloads\Julian3hybrid] C:\FragPipePlus\FragPipePlus-23.2-build3\jre\bin\java.exe -jar -Dfile.encoding=UTF-8 -Xmx110G C:\FragPipePlus\FragPipePlus-23.2-build3\tools\MSFragger-4.3\MSFragger-Commercial-4.3.jar C:\Users\Thermo\Downloads\Julian3hybrid\fragger.params --license C:\FragPipePlus\FragPipePlus-23.2-build3\license.dat C:\Users\Thermo\Downloads\250821_IO25_DDA_HCD-pd-EThcD_OGlcNc_MsBrain_01.raw MSFragger version MSFragger-Commercial-4.3 Batmass-IO version 1.35.4 timsdata library version timsdata-2-21-0-4 (c) University of Michigan RawFileReader reading tool. Copyright (c) 2016 by Thermo Fisher Scientific, Inc. All rights reserved. timdTOF .d reading tool. Copyright (c) 2022 by Bruker Daltonics GmbH & Co. KG. All rights reserved. System OS: Windows 11, Architecture: AMD64 Java Info: 17.0.10, OpenJDK 64-Bit Server VM, Eclipse Adoptium The license (C:\FragPipePlus\FragPipePlus-23.2-build3\license.dat) is valid. Customer: ThermoFisher-SanJose. Mode: Site. Expiry date: 2026-04-11. JVM started with 110 GB memory Checking database... Checking spectral files... 250821_IO25_DDA_HCD-pd-EThcD_OGlcNc_MsBrain_01.raw: Scans = 37559; MS2 ITMS = false; MS2 FTMS = true; MS2 ASTMS = false; MS1 ITMS = false; Isolation sizes = [1.6]; Instrument = Orbitrap Excedion Pro ***********************************FIRST SEARCH************************************ Parameters: num_threads = 47 database_name = C:\Users\Thermo\Downloads\2025-10-30-decoys-contam-uniprotkb_taxonomy_id_10088_AND_reviewe_2024_11_28.fasta.fas decoy_prefix = rev_ precursor_mass_lower = -20.0 precursor_mass_upper = 20.0 precursor_mass_units = 1 data_type = 0 precursor_true_tolerance = 20.0 precursor_true_units = 1 fragment_mass_tolerance = 20.0 fragment_mass_units = 1 calibrate_mass = 2 use_all_mods_in_first_search = 0 write_calibrated_mzml = 1 write_uncalibrated_mzml = 1 write_mzbin_all = 0 isotope_error = 0/1 mass_offsets = 0 use_detailed_offsets = 0 labile_search_mode = OFF restrict_deltamass_to = all precursor_mass_mode = CORRECTED localize_delta_mass = 0 delta_mass_exclude_ranges = (-1.5,3.5) fragment_ion_series = b,y,c,z, ion_series_definitions = search_enzyme_name = stricttrypsin search_enzyme_sense_1 = C search_enzyme_cut_1 = KR search_enzyme_nocut_1 = allowed_missed_cleavage_1 = 2 num_enzyme_termini = 2 clip_nTerm_M = 1 allow_multiple_variable_mods_on_residue = 0 max_variable_mods_per_peptide = 3 max_variable_mods_combinations = 5000 output_format = tsv_pepxml output_report_topN = 1 output_max_expect = 50.0 report_alternative_proteins = 0 override_charge = 0 precursor_charge_low = 2 precursor_charge_high = 3 digest_min_length = 7 digest_max_length = 50 digest_mass_range_low = 400.0 digest_mass_range_high = 5000.0 max_fragment_charge = 1 deisotope = 1 deneutralloss = 1 track_zero_topN = 0 zero_bin_accept_expect = 0.0 zero_bin_mult_expect = 1.0 minimum_peaks = 15 use_topN_peaks = 300 minIonsScoring = 2 min_matched_fragments = 4 minimum_ratio = 0.0 intensity_transform = 0 activation_types = all analyzer_types = all group_variable = 0 require_precursor = 1 reuse_dia_fragment_peaks = 0 remove_precursor_peak = 2 remove_precursor_range = -1.500000,1.500000 clear_mz_range_low = 0.0 clear_mz_range_high = 0.0 excluded_scan_list_file = mass_diff_to_variable_mod = 0 min_sequence_matches = 2 check_spectral_files = 1 variable_mod_01 = 15.9949 M 3 variable_mod_02 = 42.0106 [^ 1 add_A_alanine = 0.0 add_B_user_amino_acid = 0.0 add_C_cysteine = 57.02146 add_Cterm_peptide = 0.0 add_Cterm_protein = 0.0 add_D_aspartic_acid = 0.0 add_E_glutamic_acid = 0.0 add_F_phenylalanine = 0.0 add_G_glycine = 0.0 add_H_histidine = 0.0 add_I_isoleucine = 0.0 add_J_user_amino_acid = 0.0 add_K_lysine = 0.0 add_L_leucine = 0.0 add_M_methionine = 0.0 add_N_asparagine = 0.0 add_Nterm_peptide = 0.0 add_Nterm_protein = 0.0 add_O_user_amino_acid = 0.0 # O = pyrrolysine (237.14773 Da) add_P_proline = 0.0 add_Q_glutamine = 0.0 add_R_arginine = 0.0 add_S_serine = 0.0 add_T_threonine = 0.0 add_U_user_amino_acid = 0.0 # U = selenocysteine (150.95363 Da) add_V_valine = 0.0 add_W_tryptophan = 0.0 add_X_user_amino_acid = 0.0 add_Y_tyrosine = 0.0 add_Z_user_amino_acid = 0.0 Selected fragment index width 0.10 Da. 575080828 fragments to be searched in 1 slices (5.36 GB total) Operating on slice 1 of 1: Fragment index slice generated in 5.95 s 001. 250821_IO25_DDA_HCD-pd-EThcD_OGlcNc_MsBrain_01.raw 11.5 s | deisotoping 1.3 s [progress: 37559/37559 (100%) - 11238 spectra/s] 3.3s postprocessing 250821_IO25_DDA_HCD-pd-EThcD_OGlcNc_MsBrain_01.raw 0.3 s ***************************FIRST SEARCH DONE IN 0.464 MIN************************** *********************MASS CALIBRATION AND PARAMETER OPTIMIZATION******************* -----|---------------|---------------|---------------|--------------- | MS1 (Old) | MS1 (New) | MS2 (Old) | MS2 (New) -----|---------------|---------------|---------------|--------------- Run | Median MAD | Median MAD | Median MAD | Median MAD 001 | 0.20 0.97 | -0.03 0.82 | 1.58 4.05 | -0.58 4.03 -----|---------------|---------------|---------------|--------------- Finding the optimal parameters: -------|-------|-------|-------|-------|-------|-------|-------|------- MS2 | 5 | 7 | 10 | 15 | 20 | 25 | 30 | 50 -------|-------|-------|-------|-------|-------|-------|-------|------- Count | skip | skip | 10017| 10206| 9823| skip rest -------|-------|-------|-------|-------|-------|-------|-------|------- -------|-------|-------|-------|-------|-------|------- Peaks | 300_0 | 200_0 | 175_0 | 150_1 | 125_1 | 100_1 -------|-------|-------|-------|-------|-------|------- Count | 10206| 10187| 10203| 10115| 10108| 10233 -------|-------|-------|-------|-------|-------|------- -------|------- Int. | 1 -------|------- Count | 9957 -------|------- New fragment_mass_tolerance = 15.000000 PPM New use_topN_peaks = 100 New minimum_ratio = 0.010000 New intensity_transform = 0 New remove_precursor_peak = 2 New precursor_true_tolerance = 4.000000 PPM ************MASS CALIBRATION AND PARAMETER OPTIMIZATION DONE IN 1.374 MIN********* ************************************MAIN SEARCH************************************ Checking database... Parameters: num_threads = 47 database_name = C:\Users\Thermo\Downloads\2025-10-30-decoys-contam-uniprotkb_taxonomy_id_10088_AND_reviewe_2024_11_28.fasta.fas decoy_prefix = rev_ precursor_mass_lower = -20.0 precursor_mass_upper = 20.0 precursor_mass_units = 1 data_type = 0 precursor_true_tolerance = 4.0 precursor_true_units = 1 fragment_mass_tolerance = 15.0 fragment_mass_units = 1 calibrate_mass = 2 use_all_mods_in_first_search = 0 write_calibrated_mzml = 1 write_uncalibrated_mzml = 1 write_mzbin_all = 0 isotope_error = 0/1/2 mass_offsets = 0.0/1021.35986/1062.3864/1095.3966/1103.413/1136.4232/1150.4025/1167.4177/1177.4497/1191.429/1224.4392/1265.4657/1298.476/1312.4553/1339.5026/1353.4818/1370.4971/1386.4921/1394.5083/1427.5186/1441.4978/1458.5132/1460.5288/1468.5452/1482.5244/1501.5553/1515.5347/1532.5499/1542.5819/1556.5612/1573.5765/1589.5714/1603.5507/1630.598/1644.5773/1661.5925/1663.6082/1677.5874/1685.6038/1704.6348/1718.614/1735.6293/1751.6243/1759.6405/1776.6559/1792.6508/1806.6301/1823.6454/1825.661/1833.6774/1847.6566/1864.6719/1866.6876/1880.6669/1894.6461/1897.6821/1907.7141/1921.6934/1935.6726/1938.7087/1952.688/1954.7036/1968.6829/1976.6992/1995.7301/2009.7095/2026.7247/2028.7404/203.07938/2042.7196/2050.736/2067.7512/2069.7668/2083.746/2097.7256/2100.7615/2114.7407/2116.7563/2124.7727/2138.752/2141.788/2155.7673/2157.783/2171.7622/2188.7776/2190.7932/2198.8096/2212.7888/2229.8042/2231.8198/2245.799/2259.7783/2262.8145/2272.8464/2286.8257/2300.805/2303.8408/2317.82/2319.836/2333.8152/2334.8354/2341.8313/2358.8467/2360.8623/2374.8416/2388.821/2391.857/2393.8726/2405.8362/2407.8518/2415.8682/2429.8474/2432.8835/2434.8992/2446.8628/2448.8784/2462.8577/2465.8938/2479.873/2481.8887/2489.905/2503.8843/2506.9202/2520.8997/2522.9153/2536.8945/2537.9148/2550.8738/2553.9097/2555.9255/2563.9417/2577.921/2591.9004/2594.9363/2596.952/2608.9155/2610.9312/2624.9104/2625.931/2627.9465/2649.9421/2651.9578/2665.937/2668.9731/2682.9524/2684.968/2698.9473/2699.9678/2706.9636/2723.979/2725.9946/2739.9739/2740.9941/2753.9531/2756.9893/2759.005/2770.9685/2772.9841/2781.0005/2794.9797/2798.0156/2811.9949/2814.0107/2827.99/2829.0103/2831.026/2841.9692/2845.0051/2847.0208/2855.037/2869.0164/2872.0525/2882.9958/2886.0317/2888.0474/2900.011/2902.0266/2903.047/2916.0059/2917.0264/2919.042/2921.0576/2941.0376/2943.0532/2957.0325/2960.0686/2974.0479/2976.0635/2990.0427/2991.0632/2993.0789/365.1322/406.15875/494.1748/568.21155/609.2381/656.2276/697.25415/730.2644/771.29095/812.3175/859.307/900.33356/933.3438/947.32306/974.3703/988.3496 use_detailed_offsets = 0 labile_search_mode = LABILE restrict_deltamass_to = ST- precursor_mass_mode = CORRECTED localize_delta_mass = 1 delta_mass_exclude_ranges = (-1.5,3.5) fragment_ion_series = b,y,c,z, ion_series_definitions = diagnostic_intensity_filter = 0.03 Y_type_masses = 0/203.07937/365.1322/406.15874/568.21156/730.26438/892.3172/349.137279 diagnostic_fragments = 204.086646/186.076086/168.065526/366.139466/144.0656/138.055/512.197375/292.1026925/274.0921325/657.2349/243.026426/405.079246/485.045576/308.09761 search_enzyme_name = stricttrypsin search_enzyme_sense_1 = C search_enzyme_cut_1 = KR search_enzyme_nocut_1 = allowed_missed_cleavage_1 = 2 num_enzyme_termini = 2 clip_nTerm_M = 1 allow_multiple_variable_mods_on_residue = 0 max_variable_mods_per_peptide = 3 max_variable_mods_combinations = 5000 output_format = tsv_pepxml output_report_topN = 1 output_max_expect = 50.0 report_alternative_proteins = 1 override_charge = 0 precursor_charge_low = 1 precursor_charge_high = 4 digest_min_length = 7 digest_max_length = 50 digest_mass_range_low = 400.0 digest_mass_range_high = 5000.0 max_fragment_charge = 1 deisotope = 1 deneutralloss = 1 track_zero_topN = 0 zero_bin_accept_expect = 0.0 zero_bin_mult_expect = 1.0 minimum_peaks = 15 use_topN_peaks = 100 minIonsScoring = 2 min_matched_fragments = 4 minimum_ratio = 0.01 intensity_transform = 0 activation_types = all analyzer_types = all group_variable = 0 require_precursor = 1 reuse_dia_fragment_peaks = 0 remove_precursor_peak = 2 remove_precursor_range = -1.500000,1.500000 clear_mz_range_low = 0.0 clear_mz_range_high = 0.0 excluded_scan_list_file = mass_diff_to_variable_mod = 0 min_sequence_matches = 2 check_spectral_files = 1 variable_mod_01 = 15.9949 M 3 variable_mod_02 = 42.0106 [^ 1 add_A_alanine = 0.0 add_B_user_amino_acid = 0.0 add_C_cysteine = 57.02146 add_Cterm_peptide = 0.0 add_Cterm_protein = 0.0 add_D_aspartic_acid = 0.0 add_E_glutamic_acid = 0.0 add_F_phenylalanine = 0.0 add_G_glycine = 0.0 add_H_histidine = 0.0 add_I_isoleucine = 0.0 add_J_user_amino_acid = 0.0 add_K_lysine = 0.0 add_L_leucine = 0.0 add_M_methionine = 0.0 add_N_asparagine = 0.0 add_Nterm_peptide = 0.0 add_Nterm_protein = 0.0 add_O_user_amino_acid = 0.0 # O = pyrrolysine (237.14773 Da) add_P_proline = 0.0 add_Q_glutamine = 0.0 add_R_arginine = 0.0 add_S_serine = 0.0 add_T_threonine = 0.0 add_U_user_amino_acid = 0.0 # U = selenocysteine (150.95363 Da) add_V_valine = 0.0 add_W_tryptophan = 0.0 add_X_user_amino_acid = 0.0 add_Y_tyrosine = 0.0 add_Z_user_amino_acid = 0.0 Selected fragment index width 0.08 Da. 1150161656 fragments to be searched in 1 slices (10.71 GB total) Operating on slice 1 of 1: Fragment index slice generated in 7.97 s 001. 250821_IO25_DDA_HCD-pd-EThcD_OGlcNc_MsBrain_01.mzBIN_calibrated 0.1 s [progress: 37559/37559 (100%) - 888 spectra/s] 42.3s postprocessing 250821_IO25_DDA_HCD-pd-EThcD_OGlcNc_MsBrain_01.mzBIN_calibrated 5.6 s ***************************MAIN SEARCH DONE IN 0.982 MIN*************************** *******************************TOTAL TIME 2.819 MIN******************************** Process 'MSFragger' finished, exit code: 0 MSFragger move pepxml C:\FragPipePlus\FragPipePlus-23.2-build3\jre\bin\java.exe -cp C:\FragPipePlus\FragPipePlus-23.2-build3\lib\fragpipe-23.2-build3.jar;/C:/FragPipePlus/FragPipePlus-23.2-build3/lib/batmass-io-1.35.5.jar org.nesvilab.utils.FileMove --no-err C:\Users\Thermo\Downloads\250821_IO25_DDA_HCD-pd-EThcD_OGlcNc_MsBrain_01.pepXML C:\Users\Thermo\Downloads\Julian3hybrid\250821_IO25_DDA_HCD-pd-EThcD_OGlcNc_MsBrain_01.pepXML Process 'MSFragger move pepxml' finished, exit code: 0 MSFragger move tsv C:\FragPipePlus\FragPipePlus-23.2-build3\jre\bin\java.exe -cp C:\FragPipePlus\FragPipePlus-23.2-build3\lib\fragpipe-23.2-build3.jar;/C:/FragPipePlus/FragPipePlus-23.2-build3/lib/batmass-io-1.35.5.jar org.nesvilab.utils.FileMove --no-err C:\Users\Thermo\Downloads\250821_IO25_DDA_HCD-pd-EThcD_OGlcNc_MsBrain_01.tsv C:\Users\Thermo\Downloads\Julian3hybrid\250821_IO25_DDA_HCD-pd-EThcD_OGlcNc_MsBrain_01.tsv Process 'MSFragger move tsv' finished, exit code: 0 PeptideProphet [Work dir: C:\Users\Thermo\Downloads\Julian3hybrid] C:\FragPipePlus\FragPipePlus-23.2-build3\tools\Philosopher\philosopher-v5.1.2.exe peptideprophet --nonparam --expectscore --decoyprobs --masswidth 4000.0 --decoy rev_ --database C:\Users\Thermo\Downloads\2025-10-30-decoys-contam-uniprotkb_taxonomy_id_10088_AND_reviewe_2024_11_28.fasta.fas --combine 250821_IO25_DDA_HCD-pd-EThcD_OGlcNc_MsBrain_01.pepXML Please wait. PeptideProphet is running. It can take a long time. time="07:56:39" level=info msg="Executing PeptideProphet v5.1.2" Unknown file type. No file loaded.C:\Users\Thermo\Downloads\Julian3hybrid/250821_IO25_DDA_HCD-pd-EThcD_OGlcNc_MsBrain_01.raw Unknown file type. No file loaded.C:\Users\Thermo\Downloads\Julian3hybrid/250821_IO25_DDA_HCD-pd-EThcD_OGlcNc_MsBrain_01.raw file 1: C:\Users\Thermo\Downloads\Julian3hybrid\250821_IO25_DDA_HCD-pd-EThcD_OGlcNc_MsBrain_01.pepXML processed altogether 30232 results INFO: Results written to file: C:\Users\Thermo\Downloads\Julian3hybrid\interact.pep.xml - C:\Users\Thermo\Downloads\Julian3hybrid\interact.pep.xml - Building Commentz-Walter keyword tree... - Searching the tree... - Linking duplicate entries... - Printing results... Using Decoy Label "rev_". Decoy Probabilities will be reported. Using non-parametric distributions (X! Tandem) (using Tandem's expectation score for modeling) using search_offsets in mass mixture distr: 0.0/1021.35986/1062.3864/1095.3966/1103.413/1136.4232/1150.4025/1167.4177/1177.4497/1191.429/1224.4392/1265.4657/1298.476/1312.4553/1339.5026/1353.4818/1370.4971/1386.4921/1394.5083/1427.5186/1441.4978/1458.5132/1460.5288/1468.5452/1482.5244/1501.5553/1515.5347/1532.5499/1542.5819/1556.5612/1573.5765/1589.5714/1603.5507/1630.598/1644.5773/1661.5925/1663.6082/1677.5874/1685.6038/1704.6348/1718.614/1735.6293/1751.6243/1759.6405/1776.6559/1792.6508/1806.6301/1823.6454/1825.661/1833.6774/1847.6566/1864.6719/1866.6876/1880.6669/1894.6461/1897.6821/1907.7141/1921.6934/1935.6726/1938.7087/1952.688/1954.7036/1968.6829/1976.6992/1995.7301/2009.7095/2026.7247/2028.7404/203.07938/2042.7196/2050.736/2067.7512/2069.7668/2083.746/2097.7256/2100.7615/2114.7407/2116.7563/2124.7727/2138.752/2141.788/2155.7673/2157.783/2171.7622/2188.7776/2190.7932/2198.8096/2212.7888/2229.8042/2231.8198/2245.799/2259.7783/2262.8145/2272.8464/2286.8257/2300.805/2303.8408/2317.82/2319.836/2333.8152/2334.8354/2341.8313/2358.8467/2360.8623/2374.8416/2388.821/2391.857/2393.8726/2405.8362/2407.8518/2415.8682/2429.8474/2432.8835/2434.8992/2446.8628/2448.8784/2462.8577/2465.8938/2479.873/2481.8887/2489.905/2503.8843/2506.9202/2520.8997/2522.9153/2536.8945/2537.9148/2550.8738/2553.9097/2555.9255/2563.9417/2577.921/2591.9004/2594.9363/2596.952/2608.9155/2610.9312/2624.9104/2625.931/2627.9465/2649.9421/2651.9578/2665.937/2668.9731/2682.9524/2684.968/2698.9473/2699.9678/2706.9636/2723.979/2725.9946/2739.9739/2740.9941/2753.9531/2756.9893/2759.005/2770.9685/2772.9841/2781.0005/2794.9797/2798.0156/2811.9949/2814.0107/2827.99/2829.0103/2831.026/2841.9692/2845.0051/2847.0208/2855.037/2869.0164/2872.0525/2882.9958/2886.0317/2888.0474/2900.011/2902.0266/2903.047/2916.0059/2917.0264/2919.042/2921.0576/2941.0376/2943.0532/2957.0325/2960.0686/2974.0479/2976.0635/2990.0427/2991.0632/2993.0789/365.1322/406.15875/494.1748/568.21155/609.2381/656.2276/697.25415/730.2644/771.29095/812.3175/859.307/900.33356/933.3438/947.32306/974.3703/988.3496 init with X! Tandem stricttrypsin PeptideProphet (TPP v5.2.1-dev Flammagenitus, Build 201906281613-exported (Windows_NT-x86_64)) ***@***.*** read in 0 1+, 16977 2+, 9560 3+, 2880 4+, 669 5+, 146 6+, and 0 7+ spectra. Found 8076 Decoys, and 22156 Non-Decoys MS Instrument info: Manufacturer: UNKNOWN, Model: UNKNOWN, Ionization: UNKNOWN, Analyzer: UNKNOWN, Detector: UNKNOWN INFO: Processing standard MixtureModel ... Initialising statistical models ... Iterations: .........10.........20....WARNING: Mixture model quality test failed for charge (1+). .. WARNING: Mixture model quality test failed for charge (7+). model complete after 27 iterations time="07:57:15" level=info msg=Done Process 'PeptideProphet' finished, exit code: 0 Rewrite pepxml [Work dir: C:\Users\Thermo\Downloads\Julian3hybrid] C:\FragPipePlus\FragPipePlus-23.2-build3\jre\bin\java.exe -cp C:\FragPipePlus\FragPipePlus-23.2-build3\lib/* org.nesvilab.fragpipe.util.RewritePepxml C:\Users\Thermo\Downloads\Julian3hybrid\interact.pep.xml C:\Users\Thermo\Downloads\250821_IO25_DDA_HCD-pd-EThcD_OGlcNc_MsBrain_01_calibrated.mzML Fixing pepxml: C:\Users\Thermo\Downloads\Julian3hybrid\interact.pep.xml Writing output to: C:\Users\Thermo\Downloads\Julian3hybrid\interact.pep.xml9683336229357859353.temp-rewrite Deleting file: C:\Users\Thermo\Downloads\Julian3hybrid\interact.pep.xml Moving rewritten file to original location: [C:\Users\Thermo\Downloads\Julian3hybrid\interact.pep.xml9683336229357859353.temp-rewrite] -> [C:\Users\Thermo\Downloads\Julian3hybrid\interact.pep.xml] Process 'Rewrite pepxml' finished, exit code: 0 ProteinProphet [Work dir: C:\Users\Thermo\Downloads\Julian3hybrid] C:\FragPipePlus\FragPipePlus-23.2-build3\tools\Philosopher\philosopher-v5.1.2.exe proteinprophet --maxppmdiff 2000000 --output combined C:\Users\Thermo\Downloads\Julian3hybrid\filelist_proteinprophet.txt time="07:57:21" level=info msg="Executing ProteinProphet v5.1.2" ProteinProphet (C++) by Insilicos LLC and LabKey Software, after the original Perl by A. Keller (TPP v6.0.0-rc15 Noctilucent, Build 202105101442-exported (Windows_NT-x86_64)) (no FPKM) (no groups) (using degen pep info) Reading in C:\Users\Thermo\Downloads\Julian3hybrid\interact.pep.xml... ...read in 0 1+, 8155 2+, 4510 3+, 928 4+, 170 5+, 24 6+, 0 7+ spectra with min prob 0.05 Initializing 9027 peptide weights: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100% Calculating protein lengths and molecular weights from database c:/Users/Thermo/Downloads/2025-10-30-decoys-contam-uniprotkb_taxonomy_id_10088_AND_reviewe_2024_11_28.fasta.fas .........:.........:.........:.........:.........:.........:.........:.........:.........:.........1000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........2000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........3000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........4000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........5000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........6000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........7000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........8000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........9000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........10000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........11000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........12000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........13000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........14000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........15000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........16000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........17000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........18000 .........:.........:.........:.........:.........:.........:.........:.........:.........:....WARNING: Found the following zero-mass residues in protein entry sp|O70325|GPX4_MOUSE : U MSWGRLSRLLKPALLCGALAAPGLAGTMCASRDDWRCARSMHEFSAKDIDGHMVCLDKYRGFVCIVTNVASQUGKTDVNYTQLVDLHARYAECGLRILAFPCNQFGRQEPGSNQEIKEFAAGYNVKFDMYSKICVNGDDAHPLWKWMKVQPKGRGMLGNAIKWNFTKFLIDKNGCVVKRYGPMEEPQVIEKDLPCYL .....19000 .........:.........:.........:.........:.........:.........:.........:.........:.........:..WARNING: Found the following zero-mass residues in protein entry sp|P11352|GPX1_MOUSE : U MCAARLSAAAQSTVYAFSARPLTGGEPVSLGSLRGKVLLIENVASLUGTTIRDYTEMNDLQKRLGPRGLVVLGFPCNQFGHQENGKNEEILNSLKYVRPGGGFEPNFTLFEKCEVNGEKAHPLFTFLRNALPTPSDDPTALMTDPKYIIWSPVCRNDIAWNFEKFLVGPDGVPVRRYSRRFRTIDIEPDIETLLSQQSGNS .......20000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........21000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........22000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........23000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........24000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........25000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........26000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........27000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........28000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........29000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........30000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........31000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........32000 .........:.........:.........:.........:.........:.........:.........:.........:.........:.........33000 .........:.........:.........:.........:.........:.........:.........:......WARNING: Found the following zero-mass residues in protein entry sp|Q9JMH6|TRXR1_MOUSE : U MPVDDCWLYFPASRGRTFVQTVWVAPTCPNCCWFPGFLPPVPRPPHVPRVLLRGPRGAVLPASRPSKTLPSSSQTPCPTDPCICPPPSTPDSRQEKNTQSELPNKKGQLQKLPTMNGSKDPPGSYDFDLIIIGGGSGGLAAAKEAAKFDKKVLVLDFVTPTPLGTRWGLGGTCVNVGCIPKKLMHQAALLGQALKDSRNYGWKVEDTVKHDWEKMTESVQSHIGSLNWGYRVALREKKVVYENAYGRFIGPHRIVATNNKGKEKIYSAERFLIATGERPRYLGIPGDKEYCISSDDLFSLPYCPGKTLVVGASYVALECAGFLAGIGLDVTVMVRSILLRGFDQDMANKIGEHMEEHGIKFIRQFVPTKIEQIEAGTPGRLRVTAQSTNSEETIEGEFNTVLLAVGRDSCTRTIGLETVGVKINEKTGKIPVTDEEQTNVPYIYAIGDILEGKLELTPVAIQAGRLLAQRLYGGSNVKCDYDNVPTTVFTPLEYGCCGLSEEKAVEKFGEENIEVYHSFFWPLEWTVPSRDNNKCYAKIICNLKDDERVVGFHVLGPNAGEVTQGFAAALKCGLTKQQLDSTIGIHPVCAEIFTTLSVTKRSGGDILQSGCUG ...:.........:.........34000 .........:.........:.........:.........:.........:.........:.........:.........:. Total: 34814 Computing degenerate peptides for 3728 proteins: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100% Computing probabilities for 3927 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100% Computing probabilities for 3927 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100% Computing probabilities for 3927 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100% Computing probabilities for 3927 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100% Computing probabilities for 3927 proteins. Loop 1: 0%...20%...40%...60%...80%...100% Loop 2: 0%...20%...40%...60%...80%...100% Calculating sensitivity...and error tables... INFO: mu=2.94637e-05, db_size=39405543 Computing MU for 3927 proteins: 0%...10%...20%...30%...40%...50%...60%...70%...80%...90%...100% time="07:57:24" level=info msg=Done Process 'ProteinProphet' finished, exit code: 0 PhilosopherDbAnnotate [Work dir: C:\Users\Thermo\Downloads\Julian3hybrid] C:\FragPipePlus\FragPipePlus-23.2-build3\tools\Philosopher\philosopher-v5.1.2.exe database --annotate C:\Users\Thermo\Downloads\2025-10-30-decoys-contam-uniprotkb_taxonomy_id_10088_AND_reviewe_2024_11_28.fasta.fas --prefix rev_ time="07:57:24" level=info msg="Executing Database v5.1.2" time="07:57:24" level=info msg="Annotating the database" time="07:57:25" level=info msg=Done Process 'PhilosopherDbAnnotate' finished, exit code: 0 PhilosopherFilter [Work dir: C:\Users\Thermo\Downloads\Julian3hybrid] C:\FragPipePlus\FragPipePlus-23.2-build3\tools\Philosopher\philosopher-v5.1.2.exe filter --sequential --prot 0.01 --tag rev_ --pepxml C:\Users\Thermo\Downloads\Julian3hybrid --protxml C:\Users\Thermo\Downloads\Julian3hybrid\combined.prot.xml --razor time="07:57:25" level=info msg="Executing Filter v5.1.2" time="07:57:25" level=info msg="Processing peptide identification files" time="07:57:25" level=info msg="Parsing C:\\Users\\Thermo\\Downloads\\Julian3hybrid\\interact.pep.xml" time="07:57:26" level=info msg="1+ Charge profile" decoy=0 target=0 time="07:57:26" level=info msg="2+ Charge profile" decoy=345 target=7810 time="07:57:26" level=info msg="3+ Charge profile" decoy=146 target=4364 time="07:57:26" level=info msg="4+ Charge profile" decoy=45 target=883 time="07:57:26" level=info msg="5+ Charge profile" decoy=11 target=159 time="07:57:26" level=info msg="6+ Charge profile" decoy=1 target=23 time="07:57:26" level=info msg="Database search results" ions=9001 peptides=7893 psms=13787 time="07:57:26" level=info msg="Converged to 1.00 % FDR with 11920 PSMs" decoy=119 threshold=0.7218 total=12039 time="07:57:26" level=info msg="Converged to 0.99 % FDR with 6237 Peptides" decoy=62 threshold=0.8261 total=6299 time="07:57:26" level=info msg="Converged to 1.00 % FDR with 7315 Ions" decoy=73 threshold=0.8085 total=7388 time="07:57:26" level=info msg="Protein inference results" decoy=544 target=2816 time="07:57:26" level=info msg="Converged to 0.98 % FDR with 1846 Proteins" decoy=18 threshold=0.9405 total=1864 time="07:57:26" level=info msg="Applying sequential FDR estimation" ions=7312 peptides=6275 psms=11652 time="07:57:26" level=info msg="Converged to 0.19 % FDR with 11630 PSMs" decoy=22 threshold=0.7218 total=11652 time="07:57:26" level=info msg="Converged to 0.29 % FDR with 6257 Peptides" decoy=18 threshold=0.7218 total=6275 time="07:57:26" level=info msg="Converged to 0.25 % FDR with 7294 Ions" decoy=18 threshold=0.7218 total=7312 time="07:57:26" level=info msg="Post processing identifications" time="07:57:26" level=info msg="Assigning protein identifications to layers" time="07:57:28" level=info msg="Processing protein inference" time="07:57:28" level=info msg="Synchronizing PSMs and proteins" time="07:57:29" level=info msg="Final report numbers after FDR filtering, and post-processing" ions=7293 peptides=6256 proteins=1845 psms=11629 time="07:57:29" level=info msg=Saving time="07:57:29" level=info msg=Done Process 'PhilosopherFilter' finished, exit code: 0 PhilosopherReport [Work dir: C:\Users\Thermo\Downloads\Julian3hybrid] C:\FragPipePlus\FragPipePlus-23.2-build3\tools\Philosopher\philosopher-v5.1.2.exe report time="07:57:29" level=info msg="Executing Report v5.1.2" time="07:57:29" level=info msg="Creating reports" time="07:57:29" level=info msg=Done Process 'PhilosopherReport' finished, exit code: 0 PairScans C:\FragPipePlus\FragPipePlus-23.2-build3\jre\bin\java.exe -Xmx110G -Dlibs.bruker.dir=C:\FragPipePlus\FragPipePlus-23.2-build3\tools\MSFragger-4.3\ext\bruker -Dlibs.thermo.dir=C:\FragPipePlus\FragPipePlus-23.2-build3\tools\MSFragger-4.3\ext\thermo -cp C:\FragPipePlus\FragPipePlus-23.2-build3\lib\fragpipe-23.2-build3.jar;C:\FragPipePlus\FragPipePlus-23.2-build3\tools\batmass-io-1.35.5.jar org.nesvilab.fragpipe.util.Pai

[dpolasky]

The files get truncated when replying via email - can you go to the issue on Github and upload there?

Thanks,
Dan

[peteythecat1]

log_2025-10-31_07-50-53.txt

log_2025-10-31_07-57-37.txt

The file that ends in 53 is run as pair search and the one ends in 37 is run to search EthcD.

[dpolasky]

It looks like there are around 10k PSMs in each search and no errors, but I guess you're saying there aren't many O-glyco PSMs? Can you share the psm.tsv file from the paired scan search? You can look for PSMs with an entry in the "Total Glycan Composition" column to find the OPair PSMs.

Best,
Dan

[peteythecat1]

psm.tsv

Here you go. There is definitely Glycopeptides ID just because the same RAW file produces ID for Byonic and O-Pair in metamorpheus. So I am thinking for some reason the glycan mod is not being searched.

[dpolasky]

Thanks - yes, it looks like there is an issue with the scan pairing for this data, as there are a good number of potential glycoPSMs, but all are listed as having 'no paired scan.' Could you share one of the raw files with me to debug? You can upload here: https://drive.google.com/drive/folders/1tcHf4MN1QRKVBsHAbNFAk0U7s61ct9HM?usp=sharing, or send me an email directly (dpolasky at umich.edu)

Best,
Dan