The AScore program can process first-hits or synopsis files from MSGF+, SEQUEST, or X!Tandem to compute confidence scores for the position of modified residues. It is most commonly used to compute confidence scores for phosphorylated residues (phosphosites).
The output file includes the following columns
| Column | Description |
|---|---|
| Job | Analysis ID; 0 if no job number |
| Scan | Scan number of the MS/MS spectrum |
| OriginalSequence | Peptide sequence, as reported in the PSM results from the search engine |
| BestSequence | Either the original sequence if the modified residue is unchanged, or an updated sequence if the modification site has been moved |
| PeptideScore | Score of the top scoring peptide from the list of variants considered for a given scan |
| AScore | Phosphosite localization score; higher scores are better (see below for more info) |
| numSiteIonsPoss | Possible number of site determining ions in the theoretical fragmentation spectrum |
| numSiteIonsMatched | Number of experimental ions that matched the theoretical ions |
| SecondSequence | Second highest scoring sequence (the modified residue will be different than the BestSequence peptide) |
| ModInfo | Modified residue and modification symbol |
For the AScore value:
- 0 means unable to localize (too ambiguous due to too many S, T, and Y residues)
- 19 or higher indicates 99% certainty of the phosphosite localization
- 1000 means the peptide only has one phosphosite
- -1 means the peptide has no modified residues
Use the following steps to analyze results from a set of DMS analysis jobs. When retrieving the PHRP data files, you can either run AScore on all of the identifications in the first-hits or synopsis file, or you can filter the data using an MSGF cutoff (which will result in a faster AScore runtime due to fewer peptides to process)
Use Mage File Processor to retrieve the required files
- Search datasets by dataset ID: 340332, 340360, 340380, 340369, 340366, 340356, 340336, 340354, 340359, 340371, 340372, 340363
- Find *_dta.zip
- Search jobs by dataset ID: 340332, 340360, 340380, 340369, 340366, 340356, 340336, 340354, 340359, 340371, 340372, 340363
- Find *_ModSummary.txt (just need one of them)
- Search jobs by dataset ID: 340332, 340360, 340380, 340369, 340366, 340356, 340336, 340354, 340359, 340371, 340372, 340363
- Find *_fht.txt Create a single, combined file using "Process files to local folder"
- Use the "Add Job Column" mapping to assure that the Job number is the first column
- Name the file Leishmania_TMT_NiNTA_msgfdb_fht.txt
Use Mage Extractor to filter the MSGF+ results with MSGF_SpecProb < 1E-10
- Search jobs by dataset ID: 340332, 340360, 340380, 340369, 340366, 340356, 340336, 340354, 340359, 340371, 340372, 340363
- Select the MSGFPlus jobs
- Set the MSGF Cutoff to "1E-10" and Result Type to Extract to "MSGF+ Synopsis First Protein"
- Define the output file: Leishmania_TMT_NiNTA_filtered_results.txt
- Click "Extract results from Selected Jobs"
Alternatively, manually create the PHRP data files using Peptide Hit Results Processor (PHRP)
Unzip the _DTA.zip files
Copy the AScore parameter file from \\gigasax\DMS_Parameter_Files\AScore to your local computer.
Use either of these files:
- AScore_CID_0.5Da_ETD_0.5Da_HCD_0.05Da.xml
- AScore_CID_0.5Da_ETD_0.5Da_HCD_0.05Da_MSGF1E-12.xml
This file is only required if the input file has results from multiple jobs. It is a tab-delimited file with two columns, Job and Dataset
Run AScore Console, using the switches described below.
Results files will be the input file, but with _AScore.txt in the filename, for example:
- CPTAC_CompREF_00_iTRAQ_NiNTA_01b_22Mar12_Lynx_12-02-29_msgfplus_fht_AScore.txt
AScore_Console.exe is a console application, and must be run from the Windows command prompt.
-T:SearchEngineType
-F:FhtFilePath
-D:SpectrumFilePath
-JM:JobToDatasetMapFilePath
-MS:ModSummaryFilePath
-P:AScoreParameterFilePath
-O:OutputDirectoryPath
-L:LogFilePath
-noFM
-U:UpdatedFhtFileName
-Skip
-Fasta:FastaFilePath
-PD
Use -T to specify the search engine type, for example -T:msgfplus
- Allowed values for search_engine are: sequest, xtandem, inspect, msgfdb, or msgfplus
Use -F to specify the input file: first hits file (_fht.txt), synopsis file (_syn.txt), .mzid, or .mzid.gz
- See Peptide Hit Results Processor (PHRP)
- Example synopsis file: QC_Shew_13_05b_HCD_500ng_24Mar14_Tiger_14-03-04_msgfplus_syn.txt
- This file can optionally include results from multiple datasets
- In this case, the header for the first column must be Job
- Then, the first column of each row should be the job number for that row's PSM
Use -D to specify the file with spectra data.
- This can be a concatenated DTA file (_dta.txt), a .mzML file, or a .mzML.gz file
If the first hits file specified by -F includes job numbers in the first column, use -JM to specify a job to dataset map file.
- When using -JM, do not use -D
- Columns in the job to dataset map file are Job and Dataset (tab-separated)
- List the Dataset name in the second column
Use -P for the AScore parameter file
- Example file: AScore_CID_0.5Da_ETD_0.5Da_HCD_0.05Da.xml
Optionally use -O to specify the output directory
Optionally use -L to create a log file
Use -noFM to disable filtering on data in column MSGF_SpecProb.
- By default, data is filtered using the MSGFPreFilter score specified in the AScore parameter file
- For example, to filter on MSGF SpecProb 1E-12 use:
<MSGFPreFilter>1E-12</MSGFPreFilter>
Use -U to create an updated version of the input file, but with AScore columns appended to each row
Use -Skip to not re-run AScore if an existing results file already exists
Optionally use -Fasta to add Protein Data from Fasta_file to the output
When using -Fasta, use -PD to include Protein Descriptions in the output
AScore_Console.exe -T:sequest
-F:"C:\Temp\DatasetName_fht.txt"
-D:"C:\Temp\DatasetName_dta.txt"
-O:"C:\Temp"
-P:C:\Temp\DynMetOx_stat_4plex_iodo_hcd.xml
-L:LogFile.txt
AScore_Console.exe -T:msgfplus
-F:Dataset_W_S2_Fr_04_2May17_msgfplus_syn.txt
-D:Dataset_W_S2_Fr_04_2May17.mzML
-P:AScore_CID_0.5Da_ETD_0.5Da_HCD_0.05Da.xml
-U:W_S2_Fr_04_2May17_msgfplus_syn_plus_ascore.txt
-L:LogFile.txt
AScore_Console.exe -T:msgfplus
-F:"C:\Temp\Multi_Job_Results_fht.txt"
-JM:"C:\Temp\JobToDatasetNameMap.txt"
-O:"C:\Temp\"
-P:C:\Temp\DynPhos_stat_6plex_iodo_hcd.xml
-L:C:\temp\LogFile.txt
-noFM
AScore_Console.exe -T:msgfplus
-F:"C:\Temp\Multi_Job_Results_fht.txt"
-JM:"C:\Temp\JobToDatasetNameMap.txt"
-O:"C:\Temp\"
-P:C:\Temp\DynPhos_stat_6plex_iodo_hcd.xml
-L:LogFile.txt
-Fasta:C:\Temp\H_sapiens_Uniprot_SPROT_2013-09-18.fasta
-PD
Written by Josh Aldrich for the Department of Energy (PNNL, Richland, WA)
E-mail: matthew.monroe@pnnl.gov or proteomics@pnnl.gov
Website: https://omics.pnl.gov/ or https://panomics.pnnl.gov/
AScore Console is licensed under the Apache License, Version 2.0; you may not use this file except in compliance with the License. You may obtain a copy of the License at https://opensource.org/licenses/Apache-2.0