Elution Peak Settings During Project Creation
Importing Traces: Embedded and Non-Embedded Files
After dragging and dropping the raw files into the Samples Table, the Traces Table will automatically populate with any embedded traces that are in that file.
If a trace is not captured by the MS control software, it is still possible annotate this data in the Byos project. To do so, export the non-embedded traces to X, Y (intensity, time) data in a CSV format. Add this CSV file to the appropriate line on the Samples tab as shown below.
Selecting Trace Type and Trace Peak Options
Selecting the traces for deconvolution or importation to the project is simply done by checking the box to the left of the trace type under the Import Items column, and choosing the Trace Peak Option that is to the right of the Trace Type. There are several trace peak options for deconvolution that can be performed. These options consist of SingleSlice, AutoCompute, CustomSlice(s), FixedWidthSlice(s), or AutoComputeByApex. The last option, ImportOnly, allows for importation of the trace without deconvolution. More information about customizing these peak picking settings are detailed in the following section.
Edit Trace Peak Options: Customize Settings
Customization of the trace peak options can be found under the “Edit trace peak options…” button. Clicking this button opens the pop-up menu for customization to occur. For each of these following settings, the baseline may be customized for Auto or Flat. Under the Auto baseline, a smoothing width can be set. A larger smoothing value here can merge trace peaks, while a smaller value may split peaks.
Customizing Trace Peak Options: Single Slice
The SingleSlice trace peak option processes the entire sample as a single peak, rendering one deconvolved spectrum per file. This setting is particularly useful for simple samples, or for a “quick first look” at your data.
Customizing Trace Peak Options: AutoCompute
The AutoCompute trace peak option is our default peak picking algorithm. This peak picking algorithm determines the trace peaks based on their minimum width after smoothing. This setting will render a deconvolved spectrum per peak.
Customization of the smoothing width and minimum width may be needed for optimal peak picking. In the example shown below, the smoothing width setting will look for an inflection point every 0.1 minutes. The minimum width setting states that peaks will be 0.5 minutes wide. For wide, choppy, peaks these settings should be increased. Likewise, for thin, pointy peaks, these settings may need to be decreased.
Customizing Trace Peak Options: CustomSlice(s)
The CustomSlice(s) trace peak option allows for analyst defined start and end time points, rendering a deconvolved spectrum per each time point. This setting is particularly useful for IdeS digests, or samples in which peaks are expected to elute within particular time windows.
Customizing Trace Peak Options: FixedWidthSlice(s)
The FixedWidthSlice(s) trace peak option divides the trace peak(s) by a regular interval. This setting will render a deconvolved spectrum for each interval. FixedWidthSlice(s) are particularly useful for samples with poor chromatographic separation, such as AAVs. In the example below, the first slice (interval) was set from 3.25 minutes to 3.50 minutes. The step between slices was set to 0.25 minutes, or the same duration as the first slice as to not overlap the slices. Keep in mind that decreasing the step in between slices for some overlap may be beneficial for particularly complex samples. Finally, the optional
setting, Number of slices, was set to 10. This equates to 10 slices, at 0.25 minutes a piece, or slices for 2.5 minutes.
Customizing Trace Peak Options: AutoComputeByApex
The AutoComputeByApex trace peak option operates in the same way as our default (AutoCompute) peak picking algorithm. Please see the above section for peak picking customization details. This setting allows for finer control of the chromatographic time range. The two options are “that contains apex within” or “truncated by”. The “that contains apex within” will select the entire peak if a shoulder or tail falls before or after the time range indicated.
Alternatively, the “truncated by” setting will perform a hard cutoff and exclude any shoulder or tail falls before or after the time range indicated.
Edit Global Trace Peak Filters
The “Edit global trace peak filters” button on the bottom right corner of the screen allows for more control of the trace peaks.
The peak options portion, allows for an optional time cutoff. Additionally, a minimum peak area threshold can be applied to skip low abundant peaks. By default, this threshold is set to 5.00%. If you find that you are missing the low abundant peak(s) or slice(s) this filter can be adjusted to a lower percentage or set to zero.
The peak comparison options portion of the pop-up is for comparison projects. By default, any comparison trace’s peak that falls above or below the reference peak by a ratio 10 will be highlighted in red.
Elution Peak Settings Adjustments After Project Creation
Editing Elution Peaks Globally
The quickest and easiest way to adjust the elution peaks for all samples after project creation is under Edit> Recompute trace peaks and baseline… The popup for customizing the trace peaks will open for adjustment. After globally editing the trace peaks, go to Edit> Auto compute and assign masses to globally deconvolute your traces.
Editing Elution Peaks Manually
In some cases, globally adjusting the trace peaks is necessary. Above the trace peaks table, buttons to Add, Delete, Merge, and Split peaks are available. Start and end times can be edited for a peak by clicking and editing the times in the trace peaks table, or by dragging the pink bars on the trace plot chromatogram.
Editing Global Trace Peak Filters
The global trace peak filters menu can be accessed after project creation after by clicking on the magnifying glass button above the trace peaks table. Here, the optional time range, minimum peak area threshold, and comparison project ratio flagging can be edited.