The Protein Metrics Progressive Deconvolution workflow is a new intact workflow that performs deconvolution iteratively on sequential time ranges of a chromatogram which can discern co-eluting species and distinguish isomers based on their elution profile. Relative quantitation of proteoforms present in the sample can be performed by utilizing the resulting mass XICs, which provides a more comprehensive assessment of relative levels across the time range of the progressive deconvolution experiment compared to traditional trace peak deconvolution. Here we provide details that guide the user through the key aspects of Progressive Deconvolution.
Workflow Specific Parameters
Progressive Deconvolution is now a default workflow offered within Protein Metrics Byos and Byosphere v5.12 platforms. The workflow utilizes our current Intact workflow with a few new parameters detailed below.
Trace Table
In the Traces table located in the workflow Samples tab, the user can specify the first slice range, steps between slices (defining the overlapping intervals), and the number of slices. Deconvoluted mass candidates from each small slice segment provide discrete elution time profiles of the unique masses.
Overall processing time is expected to be longer than traditional intact deconvolution. We recommend starting with wider slice ranges and a smaller number of slices, then the user can fine tune the parameters to dig deeper into the data. Smaller steps between slices can resolve closely eluting species but will also increase the number of overlapping slices and overall processing times.
By default, the trace peak options in the Traces table will be set to Slices (1). In the Edit trace peak options (2) pop-up window, the user should optimize the slice settings (3). In the example below, the first slice will start at a RT of 7.0 mins. Each slice will be 0.4 minutes wide and overlap the consecutive slice by 0.2 minutes. A total of 35 slices will be integrated resulting in the last slice starting at a RT of 14.0 mins.
Note: Data with slower acquisition rates may need wider slices.
Advanced Commands
Protein Metrics Progressive Deconvolution has several unique advanced commands within the workflow that can be adjusted by the user. It is recommended to start with the default values and then adjust the resolution, number of consecutive features, and sensitivity to dig deeper into the data when needed.
- FeatureMassResolution - The mass tolerance, in Daltons, used to associate mass observations between consecutive deconvolved spectra.
- MinConsecutiveObservations - The number of times that a mass observation must be observed across deconvolved scans to be identified as a mass features. This feature helps to remove false identifications by selecting mass features that consistently replicate within the mass tolerance.
- FeatureDepthFactor - The sensitivity of Progressive Deconvolution. If set to a larger value, one should expect that more low abundant mass features will be reported and the analysis will take longer.
Project View
The Progressive Deconvolution project will contain a new Detected Molecules table (1) to provide a molecule centric view and approach to validation. Selection of individual detected molecule rows in this new table will control and update the Trace (2), MS1 (3), Mass XIC (4), and Deconvolved (5) plots. This is different than our traditional Intact projects which use the Trace Peaks and Masses table to separately update the Trace plot and MS1 / Deconvolved plots.
Note: Currently, there is no option to re-compute trace and deconvolution parameters after the project has been created. To change or update these parameters, the workflow will need to be updated and a new project created.
To turn on the Mass XIC plot window (4), select the Window dropdown from the top left of the project view and then Enable Mass XIC.
The user can also select multiple rows to overlay the plots (as seen above) and quickly see the different elution profile of the Mass XICs and the deconvoluted spectra allowing for the identification of low abundant species and isoforms. The max intensity and the AUC are provided for each reported feature within the Detected Molecules table and can be used for quantitative comparisons.
Relative Quantitation
The Protein Metrics Progressive Deconvolution report template is automatically generated and contains two new XIC AUC relative quantitation tabs.
Expected Mass – Rel XIC AUC (%)
- A pivot table that tabulates the relative XIC AUC % for the Expected Masses. Each vertical column by default will add up to 100%. Users can customize the normalization by changing the level setting.
Bar Chart – Rel XIC AUC (%)
- A stacked bar chart comparison of the relative XIC AUC % of the Expected Masses.
For additional help with customizing the Progressive Deconvolution workflow or report, please reach out to Protein Metrics support at support@proteinmetrics.com.
Please refer to Protein Metrics Progressive Deconvolution Posters to learn more about our new workflow: https://www.proteinmetrics.com/events/poster-requests
Progressive Deconvolution for Robust Intact MS Analysis
Dedicated computational methods for feature detection and quantification in large molecule mass spectrometry analyses