Generating Annotated Trace Plots (UV, TIC, and others) for Peptide Mapping Using Byos PTM and Chromatogram

Introduction

An annotated UV or TIC trace plot for peptide mapping allows analysts to visualize major peak assignments and is a useful figure for drug submissions. An example annotated UV trace plot chromatogram is shown below.

In Byos annotated trace plots are created using Byos Chromatography workflows, which are designed for the annotation and integration of trace plots including UV and TIC chromatograms.

Validate Peptides in Byos PTM and Create an In-Silico CSV

Users often start their peptide mapping analysis using Byos PTM, a solution designed for assigning peptides and PTMs and relative quantitation of PTMs (using MS1 Extracted Ion Chromatogram Area Under the Curve or XIC AUC).

Following Byos PTM automated peptide and PTM assignment, users will often inspect and validate their peptide assignments. The Byos PTM Project Investigation View is well designed for manual inspection and analyst validation of results (see figure below). Users can then export the validated Byos PTM results as an In-Silico CSV list, which is used later to direct trace peak assignment in Byos Reference Chromatography (In-Silico).

The steps below provide basic instructions for creating an In-Silico CSV list of validated peptides in Byos PTM.

1. Analyst designates all confirmed peptide assignments as True-Positive using the Validate column. Assignments determined to be false or undesired by the analyst can be designated as False-Positives.

2. Apply filtering to only include the confirmed peptide and PTM assignments.

3. Create a CSV file In-Silico list including all confirmed peptide hits.

Reference Chromatography (In-silico) Workflow Setup:

After generating a validated peptide and PTM CSV in-silico list, the Byos Reference Chromatography (In-silico) workflow is set up according to the steps below.

1. Open the default Reference Chromatography (In-silico) workflow.

2. Add datafile(s), set Digestion, and select traces. Include both the TIC and UV Trace for automatic UV and MS alignment.

3. Adjust Autocompute settings as needed for optimized automatic peak picking.

TIP: Trace peak integration can be adjusted and recomputed post-project creation with Edit>Recompute trace peaks and baseline…

4. Set the Global Filter settings to exclude solvent and column wash and set peak relative abundance limit as needed. 

5. Add protein sequence(s).

6. Set the processing nodes for an In-Silico CSV search. This will assign trace peaks based on the Byos PTM validated peptide in-silico CSV list.

7. Set Candidate matching parameters as needed. The minimum peak intensity percentage cut off is determined relative to the MS base peak in each individual trace peak's MS spectrum. 

8. Add the scan summing and time corr. filter advanced command. EnableMS1Sum will sum the MS1 signal across the integration range and TimeCorrFilterValue will reduce background noise in the spectrum.

[Byomap]
EnableMS1Sum=true
TimeCorrFilterValue=0.1

9. Link in desired report template (.rptc), UI Layout (.ini), UI Column Filters (.cft), and Trace Plot Label Script (.tes) as needed. The Label Script can can also be pasted in as text.

10. Save the workflow to create your own “default” Byos Reference Chromatography (In-silico) workflow for future use to limit workflow creation steps needed. Click Create Project… when ready.

Reference Chromatography (In-silico) Trace Peak Inspection:

Following project creation, trace peak annotation should be inspected and corrected as needed to ensure all desired trace peaks are found and annotated appropriately. Automatic trace peak picking and assignment should work well for most peaks. The AutoCompute trace peak picking settings (see step 3 in the above section) and Global Filters (see step 4 in the above section) can be adjusted if automatic trace peak picking performs poorly. Even with optimized peak picking settings, manual trace peak adjustment is usually needed for some of the trace peaks. Byos includes tools to make manual trace peak adjustment easy. See the Chromatogram Tutorial Video Series for useful tips.

Reference Chromatography (In-silico) Annotated Trace Peak Reporting:

Byos Reference Chromatography (In-silico) includes automated and customizable reporting. This section provides some tips specific to generating and reporting annotated TIC or UV Trace plots.

The Report Plots Tab reproduces Trace Plot images captured in the Project Investigation view.

If needed segments of the plot can be automatically generated and saved. 

Byos reports can be exported as a PDF, Excel workbook, or images.

In addition to Byos Reports, images and figures can be easily exported or copy-pasted into other programs from the project investigation view.