The first step of setting up an analysis depends on preference: start either on the Plates page by selecting Add Plate or on the MS Files page by selecting Link to Plate. Alternatively, you can start on the Projects page and select existing samples.

For more information on ways to set up an analysis, see Analysis setup workflows.

PAS offers an easy way for you to reanalyze previously analyzed data, which is useful when you want to select different analysis parameters on the same data. On the Analyses page, you select one or more analyses, select the Reanalyze button, and then change whatever analysis parameters you want.

For more information, see Reanalyze one or more analyses.

The Projects page offer two ways to start an analysis: Analyze and Analyze Controls. The Analyze button analyzes all selected samples in a project, including controls. The Analyze Controls button analyzes controls in the project only. Both buttons open a dialog where you can name the analysis, specify the analysis protocol, and input other general information.

For more information, see Select samples or controls to set up an analysis.

The plate map file is a .csv file that specifies the location of each sample and control in a 96-well plate. ICS generates the file and automatically populates each column except MS file name. You must specify the MS data file name in the first column of the file, and then upload it to PAS during analysis setup.

For more information, see Plate map file format.

Yes, you can set up an analysis and start it later. Start by following the instructions in Add a plate to set up an analysis or Link to plates to set up an analysis. When you reach the last tab of the dialog, clear both checkboxes. PAS saves the analysis, which you can start by going to the Projects page, selecting the analysis, and selecting Analyze. Between setting up and starting an analysis, you can rename the project, add samples, and otherwise modify the data.

For more information, see Data Management.

View changes to the Analysis page

• New Analysis Protocol

  • The new DIA-NN (1.8.1) — System Provided DIA Protocol, Ver Sep2023 analysis protocol includes a vastly improved spectral library built from DDA data collected using a ThermFisher Orbitrap Astral Mass Spectrometer and a new scalable version of match between runs (MBR) engineered by Seer’s team.

• Analysis Results Reorganization

  • The results and visualizations for each analysis have been reorganized to make it easier to quickly access biological insights. The analysis output content is now organized into the following tabs:

    • Analysis Results - aggregate results and visualizations as well as group analysis.

    • Analysis Metrics - sample by sample results as well as experiment quality control visualizations.

    • QC Charts (if applicable) - results specific to quality control samples.

  • The raw data and group analysis results tables have been replaced by a single new Tabulated Results table that provides quick access to protein- and peptide-level analysis results.

  • New export options are now available on the tabulated results table

    • the tabulated results in matrix format export file includes all data in the tabulated results table in either protein- or peptide-mode.

    • the processed intensities export file can be exported in matrix or long formats and includes raw log10 intensities as well as imputed and/or normalized log10 intensities if those we computed during group analysis.

  • The results of group analyses will now be saved automatically. The panel will display the results of the most recent group analysis calculation performed per user.

• Minor User Interface Improvements

  • Non-significant data is now displayed on the volcano plot by default. The option to hide non-significant datapoints is now available in the volcano plot display options.

  • A box plot is automatically displayed next to the volcano plot showing results from the data point with the highest -log10 adjusted p-value.

  • Buttons to display graphs are now visible at all times. The buttons for graphs that are unavailable due to data limitations or that require data selection prior to display will be gray (inactive) by default.

  • The protein groups and peptides graphs now display as bar charts by default. The line graph is no longer available.

  • Nanoparticle-level visualizations are now hidden by default but can be unhidden in the display settings.

  • The heatmap visualization is no longer available.

• New Spaces functionality

  • Spaces offer an easy way to manage and segregate data for different external clients or internal teams

  • Spaces are used to organize MS data files, VCF files, plates and samples, projects, and analyses into individual workspaces to which users can be assigned

  • Administrators can assign users to multiple Spaces, and only those Spaces (as well as a general Space) are viewable when the users log in

  • PAS tables can be filtered by Space, showing only the data for the selected Space

  • Spaces replace the User Groups feature in PAS

• Improvements to the Link to Plate setup workflow

  • Added the ability to link up to 10 plates at a time

  • Removed redundant buttons

  • Added more supporting text to the user interface

  • Added more checks throughout the workflow to notify users earlier if something is incorrect (e.g., wrong file type, missing files)

  • Made the table of files on the Link to Plate dialog larger and easier to read

  • Made other miscellaneous layout changes to the Link to Plate dialog

• Improvements to the Analyses page

  • Added a Reanalyze button to allow users to easily reanalyze previously analyzed data sets, typically with different analysis parameters (e.g., MS method, analysis protocol)

• Improvements to the VCF Files page

  • Added a new dialog for adding VCF files, making the process more intuitive

• Improvements regarding search engines

  • Added the ability for users to adjust more parameters of the DIA-NN search engine

  • PAS now accepts .mzml files for all analysis protocols

• Improvements to the Seer AutoUploader

  • Added the ability to create and manage multiple connections between local folders on the MS controller computer where MS data files are stored and destination folders in PAS Spaces to which files are uploaded automatically

  • Added a notification system for future customer alerts

• Improvements to user assistance

  • Added more tool tips in the PAS user interface

  • Updated the PAS Help system

• General improvements

  • Improved the layout of menu buttons throughout

  • Fixed an issue in the Setup Group Analysis dialog that was incorrectly assigning column headers

  • Fixed an issue with exporting a violin plot of the Intensity Distributions graph (under Analysis Metrics) when viewed in dark mode

  • Corrected the title of the Volcano graph (under Group Analysis)

  • Made other minor bug fixes

• New analysis protocol

  • Integration of MSFragger¹ version 3.5 database search engine for DDA library-based search

• Improvements to existing analysis protocols

  • Integration of the updated DIA-NN² version 1.8.1 analysis protocol for DIA library-based and library-free search

• Improvements to user assistance

  • Updated content in the PAS Help system

  • Updated tooltips in the PAS user interface

• General improvements

  • Minor user interface improvements

  • Minor bug fixes

¹Kong, Andy T., Leprevost, Felipe V., Avtonomov, Dmitry M., Mellacheruyu Dattatreya, and Nesvishskii, Alexey I. MSFragger: ultrafast and comprehensive peptide identification in mass spectrometry–based proteomics. 14 Nature Methods (April 2017): 513-520, doi:10.1038/nmeth.4256.

²Demichev, V., Messner, C.B., Vernardis, S.I. et al., “DIA-NN: neural networks and interference correction enable deep proteome coverage in high throughput.” 17 Nat Methods (January 2020): 41–44, doi.org/10.1038/s41592-019-0638-x.

• PAS Proteogenomics Workflow to identify and explore peptides with protein sequence altering genetic variants

  • Build a custom peptide database

    • New feature to build a customized peptide sequence database using user-uploaded custom or sample-specific variant call files (VCF).

      • VCF files are used to annotate genetic variants that may result in amino acid variants (i.e., variant peptides) not captured in the canonical reference database.

    • Variant peptide sequences are combined with the reference database to generate customized peptide sequence databases.

  • Search for variant peptides

    • Using the customized peptide sequence database, now users can search their DDA data for variant peptides using MSFragger.

  • Variant Peptide Browser and Proteogenomics Data Explorer

    • Variant Peptide Browser — Identified variant peptides are summarized in interactive plots and table.

      • Summary table shows all identified variant peptides and can be sorted/filtered by sample, nanoparticle, protein, variant, and allele frequency. Each variant (row) links to a view within the Proteogenomics Data Explorer.

      • The number of variant peptides found across samples, conditions, and nanoparticles are shown. The distribution of variant peptide intensity is stratified by reference and alternative alleles and homozygous/heterozygous alleles across samples, conditions, and nanoparticles.

    • Proteogenomics Data Explorer — Interactive tool to explore peptide and variant peptide data maps in genomic space for entire proteins and at nucleic acid/amino acid resolution

      • Gene structure, protein structure, domain information, and functional region information are displayed.

      • Amino acid variants within variant peptides are highlighted, including both reference and alternative alleles.

• Improvements to analysis protocols

  • Integration of MSFragger database search engine.

    • Pre-installed MSFragger-based Proteogenomics protocol (DDA, VCF required).

  • Enabled DIA-NN MBR feature¹.

• Dashboard to monitor PAS account usage

  • Track the total number of plates added, projects added, completed analyses, and data storage.

  • Added links to most recently added plates, projects, completed analyses, and data storage.

• Expansion and improvement of analysis visualizations

  • Added additional background datasets and overlay for multiple samples in waterfall plots.

  • Added ability to interact with sample correlation and similarity plots and visualize plots showing underlying data for each comparison.

  • Added option to view and compare all metrics from plate map grid across all wells and across all plates analyzed.

  • Added option to select order of comparison for differential abundance group analysis.

  • Added new interactivity with differential abundance group analysis volcano plot display.

• Changes to file and project management

  • Added feature to multi-select projects when launching analyses.

  • Added feature to collapse Analyses folders by project.

  • Added project filtering capabilities.

• Improvements to analysis stability and plot visualizations for large-scale study sizes

  • Stability and browser performance upgrades.

  • Analysis visualization and plot rendering speed improvements.

  • Streamlined analysis summary menu improves navigation between visualizations.

• Added PAS software end user license agreement (EULA)

• Changes to help content

  • Updated help system and user guide.

  • Updated tooltips.

• Other general improvements

  • Sample Description File is now stored in Data Files.

  • Minor bug fixes.

¹Demichev, V., et al. DIA-NN: Neural Networks and interference correction enable deep proteome coverage in high throughput. Nature Methods 17, 41–44 (2019).

New analyses to visualize differences in peptide/protein group intensities between samples and groups

• New interactive table containing peptide/protein group intensities across samples/groups

• New heatmap plot for visualizing peptide/protein group intensities across samples/groups

New analysis to identify peptide/protein groups with significantly different intensities between groups (e.g., healthy vs. disease samples)

• Added new “Group analysis” sections to ‘Analysis’ section

• Guided comparison setup providing data filtering, normalization, imputation, and statistical test options

• Toggle for viewing only ‘Significant Peptides/Proteins’ in dataset

• Interactive plots and tables for exploratory expression analysis of significantly regulated targets with dynamic fold-change and p-value options

• New group analysis plots to visualize peptides/protein groups with statistically different intensities

  • Volcano plot – fold-change vs. statistical significance with coloring options to highlight proteins-of-interest

  • Coverage viewer – Amino acid sequence coverage by peptides display, including PTM detection

  • Clustered heatmap – visualizing peptide/protein group intensities across samples/groups with rows (peptide/protein groups) and columns (samples) ordered using hierarchical clustering

  • Protein-protein interaction – Visualization of PPIs (STRING db) of significant proteins

  • Intensity box plots – Compare significant peptide/protein group intensities between groups

Changes to plot visualizations

• Added ability to export plot source data to .csv files

• Improved plot rendering speeds

• Added user preferences to modify plots

New analysis protocols

• Integration of DIA-NN database search engine

  • Pre-installed DIA-NN library-based protocol (DIANN - System Provided DIA Protocol)¹

  • Expanded Seer human plasma spectral library file (4,011 protein groups; 62,687 precursors)

  • Optional in silico predicted library-free protocol

Changes to results summary

• Simplified peptide and protein group results tables

  • Panel level summary

  • NP level summary

Changes to help content

• Updated user guide

• Indexed online help

• Tooltips

New data file management

• New AutoUploader application allows automatic data transfer from LC-MS system to PAS account

¹Demichev, V., et al. DIA-NN: Neural Networks and interference correction enable deep proteome coverage in high throughput. Nature Methods 17, 41–44 (2019).

• Initial release