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----
-title: NuXL
-subtitle: Protein-RNA and DNA cross-linking
-sidebar: false
----
-
-## Introduction
-
-NuXL is a novel tool for protein-RNA and DNA cross-linking studies. It is available as a
-[stand-alone tool](#installation-as-stand-alone-tool) and as a [Proteome Discoverer community node](#installation-for-proteome-discoverer) or as a [web application](#run-as-web-application) that doesn't require you to install additional software. This guide outlines the steps to install NuXL, set up your analysis, and interpret your data using the provided tools and workflows.
-
-
-
-### Requirements:
-
-- HCD data acquired on a high-resolution MS
-- Developed and tested on orbitrap instruments (including Velos, Lumos, and Astral)
-
-## Installation for Proteome Discoverer
-NuXL is currently compatible with Proteome Discoverer 3.0 and 3.1.
-
-1. **Before Installation:**
-Download the PD archive from https://github.com/timosachsenberg/OpenMS/releases/tag/NuXLPublication
-
-3. **Before Installation:**
- - Please ensure that Proteome Discoverer is closed before running the NuXL installer.
-
-4. **Manual Installation:**
- To register the NuXL nodes in ProteomeDiscoverer, files must be copied manually to the correct folder. Note: We are currently preparing an installer to automate the installation process.
-
- For PD 3.1 you need to:
- 1. Copy `PD.OpenMS.NuXLNode.dll` to `C:\Program Files\Thermo\Proteome Discoverer 3.1\Proteome Discoverer 3.1\Thermo.Magellan.Server`
- 2. Copy `PD.OpenMS.NuXLViewer.dll` to `C:\Program Files\Thermo\Proteome Discoverer 3.1\Proteome Discoverer 3.1\Thermo.Discoverer`
- 3. Copy `ZedGraph_OpenMS.dll` to `C:\Program Files\Thermo\Proteome Discoverer 3.1\Proteome Discoverer 3.1\Thermo.Discoverer`
- 4. Go to `C:\Program Files\Thermo\Proteome Discoverer 3.1\Proteome Discoverer 3.1\Tools` and copy the folder `NuXL` from the .zip.
- 5. Open PD, go to `Administration -> Manage Licenses`. Click `Scan for Missing Features` on top of the list of available licenses on the right side of the PD window.
-
- For PD 3.0 you need to:
- 1. Copy `PD.OpenMS.AdapterNodes.dll` to `C:\Program Files\Thermo\Proteome Discoverer 3.0\Proteome Discoverer 3.0\Thermo.Magellan.Server`
- 3. Copy `ZedGraph_OpenMS.dll` to `C:\Program Files\Thermo\Proteome Discoverer 3.0\Proteome Discoverer 3.0\Thermo.Discoverer`
- 4. Go to `C:\Program Files\Thermo\Proteome Discoverer 3.0\Proteome Discoverer 3.0\Tools` and copy the folder `NuXL` from the .zip.
- 5. Open PD, go to `Administration -> Manage Licenses`. Click `Scan for Missing Features` on top of the list of available licenses on the right side of the PD window.
-
-6. **Troubleshooting**
- - Sometimes the operating system blocks the execution of .dlls copied from an external source. In these cases, it is necessary to go to the folder containing the .dll, right-click on it, choose properties, and then security. The dialog offers an option to unblock the .dll from execution.
-
-Note: Coming soon: One-click installer so no manual copy is required. The installer will automatically detect your Proteome Discoverer installation directory and deploy the NuXL nodes correctly.
-
-### Setting Up Your Study
-
-1. **Create a New Study:**
- - Open Proteome Discoverer
- - Create a new study, define a root directory and study name (e.g., `NuXL Study`). If you set up your first NuXL study, leave `(empty workflow)`
-
-2. **Adding a Protein Database:**
- - Use the `Administration' -> `Maintain FASTA file` option in Proteome Discoverer to add new protein databases to your project. Once configured, you can select this database in the NuXL node of the processing workflow.
-
-### Analysis Workflows
-
-1. **Generating an analysis Template:**
- - Click on `New Analysis`. Note: Once completed, you will later find the analysis template in "Open Analysis Template".
- - In the analysis window you can set up 'processing' and 'consensus' workflows for NuXL
- - **Processing Workflow**
- - Click on Edit in the 'Processing Step' window
- - If you want to use a preconfigured workflow click `Open` and select the template of your choice.
- - For a minimal workflow, add and connect `Spectrum Files`, `Spectrum Selector`, and `NuXL` nodes. Note: You will find the NuXL node under the `Sequence Database Search` category.
-
-
-
- - Important settings in `Spectrum Selector`: set `MS Order` to `Any` to ensure MS1 spectra are not discarded.
- - Configuring NuXL:
- - By clicking on the NuXL processing node symbol in the workflow tree, you can adapt settings Note: If you click on `Show Advanced Parameter`, you will find additional parameters.
- - NuXL provides `Presets` for different crosslinking mass spectrometry protocols. For most use cases, users just need to select a preset that fits their experimental protocol. In most cases, default parameters work reasonably well but results can sometimes be improved by tweaking other parameters.
-
-
-
- - Other Parameters:
- - `Peptide identification` -> `Precursor mass tolerance` and `Fragment mass tolerance`: Adjust the fragment search mass accuracy according to your instrument.
- - `Peptide identification` -> `Static modification` and `Dynamic modification`: Note: Standard fixed modification (e.g., `Carbamidomethyl (C)`) should be specified as Dynamic if the expected amino acid can form crosslinks, as otherwise these identifications would be lost.
- - `Cross-links` -> `Length`: Depending on your nuclease digestion protocol, you might expect different lengths of nucleotide adducts. NuXL is trained and tested on nucleotide adducts of 1-4 nucleotide length. For properly digested samples we recommend lowering the length value to 2. In most cases, the default value works well.
- - `Cross-links` -> `Sequence`: If your experiment involves a specific RNA or DNA species with a known sequence, it can be useful to enter the sequence in this field. The node then only considers possible nucleotide adduct combinations, potentially reducing false positives.
- - `Peptide identification` -> `Peptide length min`: For complex proteome searches, we recommend using the value `6` as the minimum amino acid count. For complexes of few proteins, that would allow manual validation this can be further decreased.
- - `Peptide identification` -> `Peptide length max`: We recommend setting a limit of ~30 for complex proteome-wide searches.
- - `Peptide identification` -> `Missed cleavages`: If significant crosslinking is expected to occur and create missed cleavages, the value can be increased up to 3.
-
- - Advanced users:
- - Users can define their own fragmentation rules and crosslink adduct settings. Select `none` in `Presets`. You can then define `Fragment adducts` and `Modifications` in advanced parameters according to your protocol in the following format: `[target nucleotide]:[formula]` or `[precursor adduct] ->[fragment adduct formula];[name]`. NuXL will use this information to generate all chemically feasible precursor adduct and fragment adduct combinations.
- - `Cross-link identification` -> `Can cross-link`: If you expect only a single or subset of nucleotides to be amenable to crosslinking, set this up here. Nucleotides are specified by their one-letter code without a separator (e.g. UA for U and A nucleotides)
- - `Cross-link identification` -> `Cysteine adduct`: If DTT is present during UV irradiation, it can generate additional crosslinks including a 152 Da linker. For more information see 1. If needed to be identified, we recommend specifying the composition of the linker in modifications and 'Fragment adducts' instead. Set this flag to true, if the 152 adduct is expected.
- - `Cross-links` -> `Target nucleotides`: Sum formulas of the target nucleotides of the crosslink. Heavy labeled atoms are specified with parenthesis in front with the nominal mass of the respective isotope (e.g. (13)C, (15)N).
-
- - **Consensus Workflow**: Click on Edit in the 'Consensus Step' window
- - Connect an `MSF Files` node to the 'NuXL Consensus' node.
- - Set `Spectra to Store` to `All` in the MSF Files node.
-
-
-
-
-3. **Running the Workflow:**
- - Drag and drop your Thermo Scientific .raw files onto the Processing Step in the Analysis tab
- - Important: Ensure the "By File" checkbox is selected.
- - Save the template
- - Run the workflow
-
-
-
-
-### Post-Analysis
-
-You can monitor the progress of your analysis by opening `Administration` -> `Show Job Queue`.
-
-1. **Viewing Results:**
- - Result files are not exported automatically. All results are listed under the Analysis Results tab. You can view your results in Proteome Discoverer by double-clicking the result file.
-
-
-
- - Results are displayed in several tabs. You can view: `Proteins`, `PSMs and NuXL`, `MS/MS Spectrum Info`, `Input Files`, `Specialized Traces`, and `Study Information`. Depending on your consensus workflow, you might see additional tabs.
- - The `PSMs and NuXL` table provides information about all peptide and crosslink spectrum matches (PSMs and NuXLs, respectively). Key columns include `Annotated Sequence`, `Modifications`, `Protein Accessions`, `q-value` (CSM-level q-value). We recommend to filter your results for 1% CSM-level FDR (`q-value` <= 0.01).
-
-
-
-2. **Exporting Data:**
- - To export the results for further analysis in spreadsheet software (e.g., Microsoft Excel), navigate to `File -> Export -> To Microsoft Excel`.
-
-3. **Quality Assessment:**
- - You can manually validate spectra quality by viewing annotated spectra. To do so, click on the "Show Spectrum" button in the 'PSM and NuXL' tab.
- - Assess features such as peptide sequence coverage and the presence of key ions.
-
-Note: Please take a look at the Proteome Discoverer user guide for general information on Proteome Discoverer and additional nodes.
-
-## Installation as stand-alone tool
-
-NuXL is developed using the OpenMS framework. Once installed, it is accessible through the OpenNuXL tool. OpenMS offers a suite of tools for graphical interfaces and command-line operations, compatible with Windows, MacOS, and Linux platforms. This guide focuses specifically on using the graphical application on Windows.
-
-Alongside NuXL, the following core OpenMS software modules are also available and will be utilized in this tutorial. You can click on each term to access the respective documentation:
-
-TOPPAS: A workflow design and processing tool that allows for the automated analysis of mass spectrometry data.
-TOPPView: A visualization tool that enables detailed analysis and review of mass spectrometric data.
-This step-by-step guide ensures you can leverage the full capabilities of NuXL and the associated OpenMS modules efficiently.
-
-### Downloading the OpenMS installer with NuXL.
-Download the OpenMS-NuXL installer from https://github.com/timosachsenberg/OpenMS/releases/tag/NuXLPublication
-
-### Preparing the input files: Raw data conversion.
-
-NuXL is compatible with Thermo raw files and also supports files that have been converted to the mzML format using freely available tools such as ProteoWizard's msconvert. If you provide NuXL with Thermo raw files, it automatically utilizes the ThermoRawFileParser from Thermo Scientific™ for efficient data conversion. This flexibility ensures seamless integration with your existing data processing workflows.
-
-### Getting started – setting up a basic processing workflow.
-
-Advanced users have the option to run the OpenNuXL tool directly from the command line, offering precise control over the tool's functionality.
-
-For those who prefer a more user-friendly setup, TOPPAS can be utilized to create streamlined workflows.
-
-To begin, launch an empty workflow window by running the TOPPAS pipeline tool. This approach allows you to design and manage your workflows visually, making complex configurations more manageable and accessible.
-
-To build a workflow in TOPPAS, utilize the drag-and-drop functionality. You can start by selecting the required tools from the 'TOPP tools' pane on the left side of the TOPPAS window. A basic workflow typically includes the following components:
-
-- **Input Files Node**: Designate nodes for input files labeled ``.
-- **Output Files Node**: Set up nodes for output files labeled `