MultiAlign aligns multiple LC-MS datasets to one another after which LC-MS features can be matched to a database of peptides (typically an AMT tag database). The program reads deisotoped features from either DeconTools or ICR-2LS. The LC-MS/MS Accurate Mass and Time Tag database is a SQLite file, typically generated by MTDB Creator

The MultiAlign application provides advanced visualization and manipulation capabilities for LC-MS datasets acquired on high resolution mass spectrometers. Functionalities include overlaid 2D plots, alignment plots, normalizations, and basic statistical comparisons.

MultiAlign uses the LCMSWARP algorithm to align LC-MS datasets to a master list (mass tag database or a single LC-MS dataset) then consolidates features into a consensus map. These features can be matched to an AMT tag database to identify the LC-MS features. Data processing is similar to that performed with the VIPER application, but MultiAlign has the advantage of finding consensus features followed by matching to an AMT tag DB. The processing performed by MultiAlign results in fewer "missing values" vs. the results obtained when analyzing separate datasets with VIPER.

MultiAlign Rogue is the next iteration of MultiAlign. At the moment, required input files, for each dataset, are:

• Thermo Raw File
• DeconTools _isos.csv file
• DeconTools _scans.csv file

To use MultiAlign Rogue, click New Project, then define the Project file and Output Directory via the Browse Buttons. Next, select the .Raw files for your datasets using the "Add Dataset" button

• Select the .raw files for your datasts
• The DeconTools _isos.csv and _scans.csv files need to be in the same directory as the .raw files

Once the inputs are defined, the Open button will become available; click it

MultiAlign Rogue will make a .pbf file for each .raw file, showing the progress as "Loading Raw Data"

• For 1 GB .raw files, this process can take 5 to 10 minutes per .raw file

After the data loading is complete, select the Feature Finding tab (tab #2), then choose one or more datasets, and click "Load LCMS Features".

• MultiAlign Rogue will show the Status as "FindingFeatures"
  • This can take several minutes per dataset
• Once features have been found, the Status will show "PersistingFeatures" and then "FeaturesFound"

Visualize features by selecting a dataset with state "FeaturesFound" and clicking "Plot Features" at the bottom right.

Once the features have been been found, they can be aligned across datasets using tab #3, Alignment.

• Choose a baseline datasets (AMT Tag Database is not yet supported).
• Select one or more datasets at the left, then click "Align Datasets"
  • Status will appear as "Aligning". Once alignment finishes, it will show "Aligned"
  • The baseline dataset will have Baseline in the Status file
• Future: Visualize the Alignment using "Display Alignment"
• Future: Save alignment plots using "Save Alignment Plots"

Once features have been aligned to the baseline, find LC-MS Features in common to all of the datasets using tab #4, Clustering

• Choose the datasets to cluster together (typically all of them), then click "Cluster Features"
  • Status will appear as Clustering, then PersistingClusters, and finally Clustered
• View the clustered features with the "View Clusters" button at the bottom right
• Columns in the Cluster List table are:
  • Cluster ID
  • Monoisotopic Mass
  • Normalized Elution Time (NET)
  • Abundance
  • Number of datasets that are part of each cluster (#)
• Select a cluster to update the plots and the Feature List table at the bottom right
• Additional histograms are visible under the Tools menu
• To zoom the plot, click and drag the Middle mouse button
  • Alternatively, click and drag with Ctrl+Alt+Left Button
• To zoom out fully, double middle mouse click (or double Ctrl+Alt+Left click)

To export clustered feature details, you need to open the SQLite database specified originally as the Project file

• Use DB Browser for SQLite, available at https://sqlitebrowser.org/dl/
  • Choose the "Standard installer for 64-bit Windows"
• After installing, start "DB Browser", then select the .db3 Project file
• Choose the "Browse Data" tab, then in the Table Dropdown box, select T_Clusters to see the cluster info
• Details on Clusters at the dataset level are in table T_LCMS_Features

Written by Brian LaMarche, Bryson Gibbons, and Chris Wilkins for the Department of Energy (PNNL, Richland, WA)
E-mail: proteomics@pnnl.gov
Website: https://github.com/PNNL-Comp-Mass-Spec/ or https://panomics.pnnl.gov/ or https://www.pnnl.gov/integrative-omics

MultiAlign 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