AScore Console

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).

Scores

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

Usage

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)

Retrieve Files using Mage File Processor

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)

Retrieve PHRP data files (no filter)

• 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

Retrieve PHRP data files (filter on MSGF SpecProb)

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)

Prepare Files for AScore

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

Create the Job to Dataset Map file

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

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

Console Switches

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

Example command line #1

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

Example command line #2

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

Example command line #3

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

Example command line #4

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

Contacts

Written by Josh Aldrich for the Department of Energy (PNNL, Richland, WA)
E-mail: [email protected] or [email protected]
Website: https://github.com/PNNL-Comp-Mass-Spec/ or https://www.pnnl.gov/integrative-omics

License

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