Scout
Interactomics studies play a critical role in elucidating protein structures, functions, and interactions within complex cellular environments. Cross-linking mass spectrometry with cleavable cross-linking reagents (cXL-MS) has emerged as a powerful technique for large-scale interactomics analysis by identifying proximal amino acid pairs in protein samples. However, current computational cXL-MS tools face limitations in proteomic-scale studies, such as being too slow or generating excessive false positives, particularly at the protein-protein interactions level (PPIs).
Here, we present Scout, a computational methodology that enables interactomic analysis by identifying mass spectra of peptides linked with cleavable cross-linking reagents. By leveraging machine learning techniques, Scout ensures a controlled false discovery rate (FDR) at multiple levels, including cross-linked spectrum matches, residue pairs, and PPIs. Our methodology offers an efficient and accurate solution for large-scale interactomics studies, addressing the existing computational challenges.
<b>Please cite our paper:</b><br/>
Clasen, MA, et al., “Proteome-scale recombinant standards and a robust high-speed search engine to advance cross-linking MS-based interactomics”, Nature Methods, 2024.
Equipment
Hardware
- A computer with a minimum of 16 GB RAM and 4 computing cores is recommended. However, the software can take advantage of superior configurations.
Software
Data files
Input
- Scout is compatible with data files in the formats mzML, MS2, Mascot Generic Format (MGF), Bruker® .d files (Windows-only), and Thermo® RAW files.
Output
- Scout saves results in its *.scout format, in the mzIdentML 1.2 and mzIdentML 1.3 proposed by HUPO Proteomics Standard Initiative to support the identification of cross-linked peptides. We note this is able to perform complete submissions of XL-MS data to PRIDE[<a href="#lib_1">1</a>], and is therefore compatible with the PRIDE Inspector software[<a href="#lib_2">2</a>]. Additionally, the software supports exporting all CSMs, Residue Pairs and PPIs as CSV files, as well as all results to XlinkCyNET[<a href="#lib_3">3</a>] for visualization within Cytoscape[<a href="#lib_4">4</a>].
Procedures
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Software installation
1.1 Download Scout by clicking on <i>Scout_setup_64bit.msi</i> in the latest release.
<br/>1.2 Install it according to the OS:
<br/> 1.2.1 Windows: by double-clicking in the previous downloaded file (*.msi).
<br/> 1.2.2 Linux or macOS: by uncompressing the previous downloaded file (*.zip)
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Workflow
The following workflow demonstrates how to perform a search using Scout.<br/>If you are interested to run the software in Automation mode, go to <a href="#ref_2_6">2.6</a></i>.<br/>
PS: Linux and macOS versions only work in Automation mode.
<br/><br/>
2.1 (Windows-only) Launch <i>Scout</i>: Open the Scout application to access its main window, as shown in <b>Figure 1</b>.<br/>
<p align="center"><img width="55%" alt="image" src="https://github.com/user-attachments/assets/c4bd6ee3-5585-43f3-a8d8-ba1a0ef0f989"><br/>
<b>Figure 1: Graphical User Interface of Scout’s main window.</b></p>
2.2 <b>Initial Setup</b><br/>
 2.2.1. Searching selected file(s): Check the ‘<i>Raw File(s)</i>’ radio button and then select at least one tandem mass spectra file (<i>e.g.</i>, MS2, MGF or Thermo® RAW).<br/>
  <i>PS: For Bruker® .d files, select the folder that contains the name of the file.</i><br/>
 2.2.2. Batch searching: Check the ‘<i>Raw folder</i>’ radio button and then specify a directory containing the tandem mass spectra files.
<div id="ref_2_2_3"> 2.2.3. <i>Fasta File</i>: Select a file containing the protein sequences. The file format must be in FASTA format, typically obtained from <a href="https://www.uniprot.org/">Uniprot</a>. <i>For instance</i>:</div>
  >protein name<br/>
  PROTEINSEQUENCE<br/>
<br/>
 2.2.4 <i>Output Folder:</i> Select a folder where the results will be saved.<br/><br/>
 ⇒ Click on <i>'Start'</i> button to initiate the search by using the default parameters. Once the search is complete, the results window will be opened (see item <i><a href="#ref_2_3">2.3</a></i>).<br/>
 ⇒ To stop the search, click on <i>'Cancel'</i> button and confirm.<br/>
 <i>PS: If for some reason the Scout closes, the search can continue from the point it was paused. To do this, just set the same parameters again and press the start button.</i><br/>
 ⇒ All procedures will be recorded in the <i>Log</i> box. To export it, go to File → Export log (or press ALT + M).<br/><br/>
 2.2.5 <b>Search Parameters</b><br/>
Search parameters can be adjusted to optimize the search process. To modify the parameters, navigate to Parameters → Search (or press ALT + S), as illustrated in <b>Figure 2a</b>, a new window will open (<b>Figure 2b</b>).<br/><br/>
<p align="center"><img width="35%" alt="image" src="https://github.com/diogobor/Scout/assets/7681148/255baf3a-aaa1-477a-8b20-eecfedbba462"><br/>
<b>Figure 2a: Search and Post Processing Parameters can be modified on Parameters menu.</b><br/><br/>
<img width="55%" alt="image" src="https://github.com/user-attachments/assets/b1bf3b9f-43ef-4a2e-b663-12b77cdd5f51"><br/>
<b>Figure 2b: Search Parameters window</b></p>
  2.2.5.1. <i>MS1 PPM Tolerance</i>: Specify the ppm error tolerance for the precursor mass.<br/>
  2.2.5.2. <i>MS2 PPM Tolerance</i>: Specify the ppm error tolerance for fragment ions.<br/>
  2.2.5.3. <i>Ion Pair PPM Tolerance</i>: Specify the ppm error tolerance for ion pair mass.<br/>
  2.2.5.4. <i>Min. Peptide Length</i>: Specify the minimum number of amino acids in each connected peptide.<br/>
  2.2.5.5. <i>Max. Peptide Length</i>: Specify the maximum number of amino acids in each connected peptide.<br/>
  2.2.5.6. <i>Min. Peptide Mass</i>: Specify the minimum peptide mass in Daltons.<br/>
  2.2.5.7. <i>Max. Peptide Mass</i>: Specify the maximum peptide mass in Daltons.<br/>
  2.2.5.8. <i>Missed Cleavages</i>: Specify the maximum missed cleavages allowed in a single peptide.<br/>
  2.2.5.9. <i>Max. Variable Mods</i>: Specify the maximum number of variable post-translational modifications in a single peptide.
<div id="ref_2_2_5_10">  2.2.5.10. <i> Enzyme</i>: Select a proteolytic enzyme for <i>in-silico</i> digestion.</div>
    2.2.5.10.1. <i> Add Enzyme</i>: Navigate to the <i>Enzymes</i> tab and click on ‘<i>Add Enzyme</i>’ button (<b>Figure 3a</b>). A new window will be opened (<b>Figure 3b</b>).<br/>
<p align="center"><img width="35%" alt="image" src="https://github.com/diogobor/Scout/assets/7681148/14ee608a-dafa-464f-be3c-d01003b3c83f"><br/>
<b>Figure 3a: Enzymes window – This tab enables the addition or removal of enzymes.</b><br/><br/>
<img width="35%" alt="image" src="https://github.com/user-attachments/assets/54deb2c1-1e81-490c-8c81-6ee6353c7bf6"><br/>
<b>Figure 3b: New Enzyme Inclusion – This window allows users to introduce a new enzyme to the existing list of enzymes.</b>
</p>
      2.2.5.10.1.1. <i>Name</i>: Specify a name for the new enzyme.<br/>
      2.2.5.10.1.2. <i>Sites</i>: Specify the amino acids at which cleavage should occur. <i>PS: The amino acids should be included without spaces, for instance, the trypsin sites should appear as KR.</i><br/>
      2.2.5.10.1.3. <i>Blocked by</i>: Specify the amino acids that will impede the cleavage. <i>PS: As in ‘Sites’, the amino acids must be typed without spaces.</i><br/>
      2.2.5.10.1.4. <i>C-Terminal</i>: Check this option if the new enzyme cleaves at the C-terminus of the peptide; otherwise, cleavage will occur at the N-terminus.<br/>
      Click on the ‘<i>Confirm</i>’ button to incorporate the new enzyme into the Enzymes table. Afterwards, return to <i><a href="#ref_2_2_5_10">2.2.5.10</a></i>.<br/>
    2.2.5.10.2 To remove an Enzyme, press ‘<i>Del</i>’ key. A confirmation message will be displayed. Confirm it to proceed.<br/><br/>
  2.2.5.11. <i>Enzyme specificity</i>: Select an enzyme specificity from the list: full specific or semi-specific.<br/>
  2.2.5.12. <i>Cleavable Reagent</i>: Select a cleavable cross-linker from the list.<br/>
    2.2.5.12.1. <i>Add Reagent</i>: Go to XL Reagents tab and click on ‘<i>Add Reagent</i>’ button (<b>Figure 4a</b>). A new window will be opened (<b>Figure 4b</b>).<br/>
<p align="center"><img width="35%" alt="image" src="https://github.com/diogobor/Scout/assets/7681148/b04ab7b4-6066-47b1-8e2c-dabb1eb326cc"><br/>
<b>Figure 4a: Chemical cross-linkers window: on this tab, new reagents can be added or removed.</b><br/><br/>
<img width="35%" alt="image"
