Summary

• Introduction to System Suitability Testing
• Experimental Notes
• Byos System Suitability Workflow Configuration
• Project Review
• System Suitability Report
• Dashboards in Byosphere

Introduction

The system suitability testing (SST) is to verify that an analytical method was suitable for its intended purpose when the analysis was done. Ideally, SST is conducted at the beginning and end of each analysis, ensuring the system’s suitability throughout the entire run. Tailored to individual methods, SST incorporates predefined acceptance criteria, such as mass errors below 10 ppm. 

Byos, with its versatile reporting function, streamlines the generation of system suitability reports for a variety of supported workflows, including peptide, intact, oligo, and glycan workflows. In this article, we showcase the seamless creation of an SST report for a peptide mapping experiment using Byos. 

While an SST report provides a snapshot of instrument performance, it lacks the ability to highlight trends. Complementing the SST report, Byosphere enables the creation of a dynamic dashboard. This dashboard serves as a comprehensive tool for monitoring and identifying potential trends across different instruments and locations. At the end of this article, we provide a brief guide on creating an insightful dashboard.

Experimental Notes

A peptide mixture (Pierce™ Peptide Retention Time Calibration Mixture, catalogue# 88321) was run on an Agilent 1290 chromatography system hyphenated to a Thermo Q Exactive™ Mass Spectrometer. Total run time per sample was 10 minutes, initiated under aqueous conditions and a linear gradient with an increasing percentage of an organic solvent was applied. In total three system suitability samples were acquired, one at the beginning, middle, and end of the run. 

Byos System Suitability Workflow

• Click the System Suitability icon to launch the workflow

• Three steps to project creation

 

System Suitability Workflow Configuration

Samples 

In the samples table for this workflow, follow the standard drag-and-drop procedure to upload files. However, note that for this workflow two additional columns are added to the sample table: Condition and Replicates. Both fields are required to group the samples for the report at the end of the analysis. Fill in the replicate number and condition into the respective fields. Alternatively, the assignment of the samples to a replicate number and condition can happen after project creation by changing the MS File Alias name as described here in this link.

Sequences

To incorporate the sequences of your analytes into the workflow, you have two options:

1. Select a Fasta File:

   • Choose a Fasta file containing the sequences of your analytes.
   • Upload the selected Fasta file into the workflow.

2. Manually Add Rows and Fill in the Form:

   • Alternatively, you can manually add rows to the workflow by selecting the appropriate option.
   • Fill in the form that pops up after clicking the respective button in the sequences section of the workflow configuration window.
   • For our example, each peptide sequence should be defined as a separate row in the form.

Figure 1. Sequence Input

Instrument Parameter and Digest Settings

As required in all Byos MS2 workflows, the Instrument and Digest settings should be appropriate for the sample. Select the correct enzyme and specificity to match the digest.  Precursor and fragment tolerances should be set according to actual instrument accuracy. If a lock mass is present in the file, do not forget to add it both here and later in the Quant (Byologic) panel in the “Advanced” section. Be sure to select the correct fragmentation type for the sample as well. For our example, tryptic digestion settings can be defined, that are fully specific with no cleavage sites since all peptides in our mixture are fully tryptic with no missed cleavages.

Modification and Glycan Input Settings

The System Suitability workflow Modification and Glycan settings are tailored for the example we show here. The peptides in this commercially available mixture are isotopically labelled with C13 and N15 at arginine and lysine residues. Hence, the modification settings contain two entries of fixed modification accounting for them. If the utilized analyte is different, change the modification and glycan input setting to ensure the identification of the species present in the sample.

Project Review

Upon completion of project execution, Byos will open an inspection tab and a second tab holding a report based on the project. Click the project tab to allow a detailed inspection of the peptide identifications, along with their associated XICs, MS2s, and all the information needed for a thorough review. 

Figure 2. Overview Inspection View

No extensive project review is required for the shown example. Some peptides can be selected to check the integrity of the project file. Potentially, a manual adjustment may be required for the XIC integration time intervals, which can be edited simultaneously since version 5.3 by keeping either the Shift or the Ctrl key pressed while simply dragging the black bars that mark the XIC boundary to a new location. Keeping the Shift key pressed all boundaries will all associated boundaries will snap to the same location. Pressing the Ctrl key on the other hand will keep the differences between the single boundaries while moving them right or left. Figure 3 and Figure 4 show the difference between pressing the Ctrl or Shift key. The grey lines are the boundary’s location prior manual adjustments.

Figure 3. Manual Adjustment of XIC Boundaries while Pressing the Shift Key

Figure 4. Manual Adjustment of XIC Boundaries while Pressing the Ctrl Key

A prerequisite to change multiple XIC boundaries it so to enable the stacked plots:

Figure 5. Enable Stacked Plots

System Suitability Report

Upon the completion of the project, the report tab will automatically open next to the inspection tab. If no modifications to the report settings were made during the workflow configuration before project creation, the default system suitability report will be displayed. However, even if the report settings were not altered initially, the user has the flexibility to switch to another report from the available presets or add a custom report if desired.

Preset reports including the default system suitability report can be accessed from the library via File > Presets > Report Presets:

Figure 6. Preset Reports

Custom reports can be loaded via File > Load from file:

Figure 7. Load Custom Reports

Note that as updates are made to the project, they will immediately yield new calculations in the project, but in the report, data must be refreshed.  To do so, in the report tab menu choose Tabs > Update tab content.

Summary Tab

The first tab of the default system suitability report serves as a summary tab including relevant information of the analysis. This tab can be customized via Edit > Current tab settings> Edit summary template. The editor that opens accept html formatting and additional data sources can be accessed via the Short Codes.

Figure 8. Summary Tab Editor

System Suitability Criteria Tabs

In each subsequent tab of the system suitability report, a consistent structure is maintained (see Figure 9). At the top is a text box with the title and a description of the system suitability criteria (1). Below this text box is a pivot table with single values for each peptide and each replicate (2). For each of these rows a function is applied, e.g. the maximum or the minimum (3). The last column indicates if this row fulfills the criteria described in the text box above. If the criteria are met, the cells are highlighted green and contain the text “Passed”. In cases the criteria are not met the cells are highlighted red and contain the text “Failed” (4).

Figure 9. System Suitability Report Structure

Note that if the value fields are not populated the most likely reason is that the replicates are not defined with the alias name (see section “System Suitability Configuration”, “Samples”). In cases the category and replicate number was defined prior project creation, drag and drop the corresponding fields in the column section.

The final report can be exported via File > Export and then a file format can be selected, such as Excel, PDF or the image format PNG.

Report Customization

To customize the report, duplicate a tab that has the right function applied like max or min via Tabs > Duplicate current tab. The title of the duplicated tab can be edited by double clicking on it. The text box above the table can be edited via Edit > Current tab settings > Edit free text.

Figure 10. Change the Content of The Text Box above the Pivot Table

The threshold for the system suitability criteria can be changed as shown in Figure 11 and through this steps: Edit > Edit dynamic columns > Edit post pivot dynamic columns (1). After clicking on the pencil icon (2), the Edit dynamic column editor will pop up. On the very top of the script a line defines the threshold (3). This line can be edited, and the accurate threshold can be set (in this example replace the number 10). After pressing the OK button (4) followed by the Finish and reload button (5) the new threshold is applied.

Figure 11. Change the Threshold for the System Suitability Criteria

For more information on how to customize reports, please see our related videos available at here or contact the Customer Success Team at:

support@proteinmetrics.com

 

Dashboards in Byosphere

Byosphere’s Deep Query Dashboards enable the user to monitor and screen the parameters used in the system suitability assessment across different instruments and locations. This can help to detect trends, facilitates the evaluation of instrument performance, and identifies variations across different locations. Another notable feature is the capability to set an alert based on a predefined threshold. This alerting system acts as an early warning mechanism empowering users to take swift action in response to critical changes. 

Dashboard Creation

Figure 12 shows how to create a dashboard after logging into the Byoshere web page. Click the gauge icon to get to the dashboard section (1). Here the user can either start with a template or create a new blank dashboard (2). In both cases a window will pop up that requires two entries: a file alias (file name) (3) and a storage location on the server (4).

Figure 12. Dashboard Creation

Dashboard Configuration

After creating a new dashboard it should instantly be open in the edit mode. On top of the page the title, project file location and global filter can be set (see Figure 13).

1. Default title is the file alias set during the dashboard creation but it can be changed here.
2. Set the location to retrieve the data from processed projects. Data can only be retrieved from project files, but not from raw files.
3. A global filter can be applied to the whole source data which impacts all visulizations in the dashboard

Figure 13. Dashboard in Edit Mode

A visualization can be added on the top right by clicking on the button “Add New Visualization”. Each visualization has four buttons on the top:

1. Duplicate,
2. Edit,
3. Filter,
4. Delete the selected visualization.

Figure 14. Visualization in Edit Mode

After clicking the Edit button, the visualization configuration window will open with four sections (see Figure 15):

1. In the Visualization Settings the visualization type can be selected and all relevant settings for the appearance can be set. The setting fields change according to the selected visualization type.
2. Global Filters and specific filters for the visualization can be changed in the Filter & Sort section.
3. SQL based - custom fields can be created in the Derived Fields section.
4. Background alerts can be set and a trigger that activates them.

Figure 15. Visualization Settings

After applying the settings, the dashboard configuration window reopens. Visualizations can be dragged across the grit. The visualization window size can be adjusted by clicking dragging on the right bottom corner. By clicking the “Publish” button on the top panel all settings will be saved.