Using SorcererScore™ For Low-Abundance Modified Peptides Detection

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As detailed in this technical article, SorcererScore™ will provide improved identification of low-abundance, modified peptides (LAMPs) for your SORCERER™ system. Sorcerer owners who are in-maintenance, please contact us to arrange installation of SorcererScore. This post will cover how to submit a search with SorcererScore enabled.

SUBMISSION

During the installation of the SorcererScore, search profiles called “Sample SorcererScore (Select Database)” and “Sample SorcererScore Phosphorylation (Select Database)” should have been created, they contain the search directives necessary to run SorcererScore. Please select that profile, then choose your own peptide database, and then add any additional search options you want like additional modifications, then submit the search.

What follows is a detailed description of the search options used in the SorcererScore Template search profiles. You may skip ahead to the ANALYSIS section if you wish, however it is required to go through these steps if you are using a FASTA database which already includes decoys as you need to change an option if that’s the case.

For your search conditions, please verify the following guidelines are followed for best results with SorcererScore:

We recommend a minimum mass search tolerance of 20 ppm be used.
ONE of the following must be done to allow for enough decoys for the script’s modeling:
- 1. ENABLE Automatic decoy peptide generation with a non-decoy peptide database selected. (NOTE: if you use TPP do not use this option as they program is not compatible with our virtual decoys).
  
  2. Use your own FASTA database that contains decoys as the peptide database. It MUST have “decoy_” at the front of the protein header in order for SorcererScore to recognize a protein as a decoy. DISABLE Automatic decoy peptide generation if your FASTA contains these decoys.
[Phosphorylation only] For phosphorylation searches, enable the following variable modifications in addition to your other modifications if any: Phosphorylation (STY), and PhosphorylationDehydration(ST, Monoisotopic Mass: -18.010565, Average Mass: -18.0153). Additionally for phosphorylation searches, under Advanced Options, enable isotope check using mass shift of 97.976799 to model beta-elimination in phosphorylated peptides.
[Non-phosphorylation searches]:Select your own variable modifications. Enable isotope check using mass shift of 1.003355 amu tolerance.
To enable SorcererScore, again under Advanced Options, enable the MUSE custom script option and enter the following text in the box (case sensitive, use lower case) depending on your mass spectrometer:
- For high fragment mass accuracy instruments, enter the following:
  - SS1901-100.03
- For low fragment mass accuracy instruments, enter the following:
  - SS1901-30.6
Under advanced options, for Results Scoring, select 2000 preliminary results. For Peptide Results select 20 peptide results in the result file.
Under advanced options, for Duplicate references, select 10 duplicate protein references to store additional proteins the peptide is found in, if any.

ANALYSIS

NOTE: If TPP and/or Scaffold are enabled, TPP will reflect the SorcererScore promoted results while Scaffold will reflect the original results. For analyzing SorcererScore results, our recommendation is to use the spreadsheet file which is described next.
Under the Results path link found in the Download results page of the completed search, if you click on that link and then navigate to the muse folder, you will find files named pepid_XXX.txt and a pdf file. The pdf file contains various plots of the SorcererScore enhanced results compared to the original search results. The txt file is a tab delimited spreadsheet file that should be opened with your spreadsheet program (ex. Excel). Here is a description of its column values:
- protein1 – This is first protein reference in the FASTA where the peptide is found.
- pepfdr – The peptide’s false discovery rate.
- sscore – The SorcererScore discriminant score.
- dfragm – This is a measure of the average (root mean square actually) of the delta of the fragment ions peaks matched compared to the theoretical.
- frags – Total number of fragment ions matched for that peptide.
- peplen – Length of the peptide.
- dmass – Delta mass of the observed precursor mass compared with the theoretical for that peptide.
- idscan – Mass spec sample and scan number for that peptide
- pep – Peptide. ^ indicates the N-term, $ indicated the C-term. Modifications are shown in () for variable modifications and {} for static modifications. Any periods seen in the peptide indicate y-ion fragment matches found at that position. The number of periods will match column frags.
- prot2etc – If the Sorcerer search advanced option of duplicate protein references was set to display 1 or more duplicate protein references the peptide is found in, those additional protein references which contain the peptide if any are listed here. The format is a number which represents the number of addition protein references that peptide is found in followed by the protein references separated by commas in between {}. 0{} indicates the peptide is only found in the protein shown in column protein1.

Using SorcererScore™ For Low-Abundance Modified Peptides Detection

SUBMISSION

For your search conditions, please verify the following guidelines are followed for best results with SorcererScore:

We recommend a minimum mass search tolerance of 20 ppm be used.

ONE of the following must be done to allow for enough decoys for the script’s modeling:

1. ENABLE Automatic decoy peptide generation with a non-decoy peptide database selected. (NOTE: if you use TPP do not use this option as they program is not compatible with our virtual decoys).

2. Use your own FASTA database that contains decoys as the peptide database. It MUST have “decoy_” at the front of the protein header in order for SorcererScore to recognize a protein as a decoy. DISABLE Automatic decoy peptide generation if your FASTA contains these decoys.

[Non-phosphorylation searches]:Select your own variable modifications. Enable isotope check using mass shift of 1.003355 amu tolerance.

To enable SorcererScore, again under Advanced Options, enable the MUSE custom script option and enter the following text in the box (case sensitive, use lower case) depending on your mass spectrometer:

For high fragment mass accuracy instruments, enter the following:

SS1901-100.03

For low fragment mass accuracy instruments, enter the following:

SS1901-30.6

Under advanced options, for Results Scoring, select 2000 preliminary results. For Peptide Results select 20 peptide results in the result file.

Under advanced options, for Duplicate references, select 10 duplicate protein references to store additional proteins the peptide is found in, if any.

ANALYSIS

NOTE: If TPP and/or Scaffold are enabled, TPP will reflect the SorcererScore promoted results while Scaffold will reflect the original results. For analyzing SorcererScore results, our recommendation is to use the spreadsheet file which is described next.

protein1 – This is first protein reference in the FASTA where the peptide is found.

pepfdr – The peptide’s false discovery rate.

sscore – The SorcererScore discriminant score.

dfragm – This is a measure of the average (root mean square actually) of the delta of the fragment ions peaks matched compared to the theoretical.

frags – Total number of fragment ions matched for that peptide.

peplen – Length of the peptide.

dmass – Delta mass of the observed precursor mass compared with the theoretical for that peptide.

idscan – Mass spec sample and scan number for that peptide

pep – Peptide. ^ indicates the N-term, $ indicated the C-term. Modifications are shown in () for variable modifications and {} for static modifications. Any periods seen in the peptide indicate y-ion fragment matches found at that position. The number of periods will match column frags.

Please email us with any comments or questions, and any suggestions to help us improve this script: info@SageNResearch.com.

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