To support the development of robust and reproducible assays, Panorama offers reporting that summarizing analysis of replicates of the same sample multiple times a day over multiple days to assess inter- and intra-day variability. 3 replicates over 3 days or 5 replicates over 5 days are common usages. As long as at least 3 runs are included, Panorama will generate the Reproducibility report for that protein. The report relies on a Replicate annotation in Skyline to identify the day for grouping purposes. The Reproducibility Report visualizes the reproducibility of the assay and which peptides perform best.

Configure Skyline Document

For this report, you will load a Skyline document containing multiple replicates for a protein. You must include a Day replicate annotation, and you may want to also captures both NormalizedArea and CalibratedArea values, in order to take advantage of the options available below.

To configure, start in Skyline:

  1. Select Settings > Document Settings
  2. On the Annotations tab, click Add...
  3. Use "Day" as the name, and leave the Editable button toggled
  4. Check Replicates from the "Applies to" list
  5. View->Document Grid and choose "Replicates" from the Reports drop-down
  6. Fill in values for the Day column for the replicates that you want to include in the reporting. You can use whatever values you like to identify the timepoint - "Day 1", "Day 2", "10-08-2021", "Batch 1", etc
Optionally, add annotations to capture the normalized and calibrated areas.
  1. Select Settings > Document Settings
  2. On the Annotations tab, click Add...
  3. Use "NormalizedArea" or "CalibratedArea" as the name, and choose the Calculated button
  4. Check Precursor Results from the Applies to list
  5. Choose the value to plug in for the annotation. Precursor->Precursor Results->Precursor Quantitation->Precursor Normalized Area or ->Calculated Concentration. Use "Sum" as the aggregate

Create Folder and Load Document

To use this report, start in a Panorama folder of type "Chromatogram library".

When you import a Skyline document containing protein data, the system will automatically add a new Proteins tab.

The protein list on this tab contains Protein/Label, File Name, Description, Protein Note/Annotations and Library State (if available). For proteins where there are multiple replicates included in the document, you will see a link next to the protein / label. Click it to open the Reproducibility Report.

View Reproducibility Report

The report will look for a Replicate annotation named "Day" in the Skyline document to analyze for intra- and inter-day variability. In the absence of this annotation, the report will not be available.

The report contains a series of panels:

  • Protein: This overview section shows some basic metadata about the protein and the file it was loaded from.
  • Sequence Coverage: View the peptide coverage within the protein sequence; highlight various features.
  • Annotations
  • Precursor: The precursor listing can be filtered and sorted to customize the report. Any CV values (inter-day, intra-day, or total) over 20 in this section are highlighted in red.
  • Peak Areas
  • Coefficient of Variation
  • Chromatograms: Clicking on a precursor in the plots or summary table will select that precursor and scroll to its chromatograms at the bottom of the page.

Below the Chromatogram panel:

  • Calibration Curve/Figures of Merit: If the imported Skyline file contains calibration data, we will show the Calibration curve on the page. Figures of Merit (FOM) will be displayed below the calibration curve.
    • The Figures of Merit section will only display the Summary table, with the title linking to the detailed FOM page
    • The Calibration Curve title is also a link that would bring the user to a separate Calibration Curve page.

Protein Heading

In the Protein panel at the top of the report, you will see information about the Skyline document, including a link to download it.

Sequence Coverage

If the Skyline document includes a protein sequence, you will see a Sequence Coverage section. The peptide coverage is shown as a shaded bar within the overall protein sequence. A set of Features checkboxes lets you highlight the protein features at each amino acid with assigned colors.

Annotations

If relevant annotations are available, they will be shown in the next panel. For example:

Precursor Listing

  • Sort by lets the user sort the precursors on the plots shown by:
    • Intensity (Default)
    • Sequence Location
    • Coefficient of Variation
  • Value type: When the document contains calibrated and/or normalized values, you can select among them. The default will depend on what's in the Skyline file: Calibrated, Normalized, and Raw in that order.
    • Calibrated
    • Normalized
    • Raw
  • Sequence Length: Drag the min/max endpoint sliders to filter by sequence length.
Note that if the report includes 1xN (a single day), the inter-day CV column will still be present, but values will be 0.0%.

Peak Areas

In the Peak Areas plot, users can compare the precursors filtered and sorted above.

In the plot, peptides are labelled using Skyline’s abbreviation. Charges from -3 to +3 are shown with that number of pluses or minuses. For example, +2 will be rendered as “++” for improved readability.

The plot will show each replicate’s peak area, divided by run within the day, and day. A box plot will show the full range of all values. Data points will be arranged from left to right within each precursor based on their acquisition time.

Hover anywhere on the plot for buttons to print to PDF or PNG.

Coefficient of Variation

Based on which Show checkboxes are checked, this plot shows the averages for intra-day and inter-day peak areas, plus the "Total CV", which is the square root of the sum of the squares of the intra and inter-day CVs.

On the CV plot, hover over individual bars for a tooltip:

  • For the inter- and intra-day CVs, see the full peptide sequence (with charge state and mz) and show the standard deviation of the CVs, as well as the average CV (which is the value being plotted).
  • For the total CV, we’ll just show the value and peptide sequence.
Hover anywhere on the plot for buttons to print to PDF or PNG.

Chromatograms

One chromatogram per replicate is rendered, in a matrix-like layout, with rows that represent the days the data was acquired and columns that represent the indices within the day. Hence, the layout will reflect the MxN nature of the data. To see the full layout, expand your browser view. Individual plots are titled based on the day and index.

  • Users can click to download a PDF or PNG of any chromatogram. Hover to see the buttons.

At the bottom of the panel of chromatograms, you can click Show peptide details to switch to the detail page with more adjustable ways to view chromatograms.

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