The AScore program can process first-hits or synopsis files created by Peptide Hit Results Processor (PHRP) to compute confidence scores for the position of modified residues, as identified by MS-GF+, MaxQuant, MSFragger, X!Tandem, etc. AScore is most commonly used to compute confidence scores for phosphorylated residues (phosphosites).

The output file includes the following columns

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 MS-GF+ 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 "MS-GF+ 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:

• Supported formats: First hits file (_fht.txt), synopsis file (_syn.txt), .mzid, or .mzid.gz
• First hits files and synopsis files are tab-delimited files created by the Peptide Hit Results Processor (PHRP)
  • Example synopsis file: QC_Shew_13_05b_HCD_500ng_24Mar14_Tiger_14-03-04_msgfplus_syn.txt
• Mzid files correspond to the mzIdentML Data Standard for Mass Spectrometry-Based Proteomics Results (PMID:22375074)
  • .mzid.gz files are gzipped .mzid files
• First hits files and synopsis files 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 is typically a .mzML file, or a .mzML.gz file
  • The program also supports the legacy concatenated DTA file (_dta.txt) format

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://github.com/PNNL-Comp-Mass-Spec/ or https://www.pnnl.gov/integrative-omics

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