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Deconvolution Parameters
The first tab for deconvolution, in yellow, is labeled UniDec Parameters. In this tab, we can set the range of charge states, the mass range, and the sample rate of the deconvolved mass spectrum in daltons (Da).
The charge range sets a range of charges that can be assigned for the m/z peaks in the mass spectrum. If we set a minimum of 10 and a maximum of 25, then UniDec cannot assign a charge state of 9 or lower, nor a charge state of 26 or higher. Picking a charge range that does not include the true charge states for the m/z peaks will result in a distorted deconvolved mass spectrum or an error message (see video: Deconvolution Parameters Part 1, for an example). It is often better to start with a wider range of charge states and then narrow the range to the charge state distribution of interest. You can also narrow the charge range to remove artifacts.
Like the charge range, the mass range sets a range of masses, in Da, that can be assigned for the m/z peaks in the spectrum. Unlike zooming into the m/z range (see data processing for more info), the mass range sets the allowed deconvolved masses for the available data. Setting this mass range lower or higher than the true masses will either create artifacts, cut off certain analytes, or give an error message. Similar to the charge range, it is often better to start with a wider range then narrow the range later. Narrowing the mass range can help remove artifacts.
The Sample Mass Every parameter sets a sample rate for the deconvolved mass spectrum. If a sample rate of 10 Da is set, then there will be a mass data point every 10 Da. Every mass data point would fall on an even 10, such that a data point would not appear at 66,417 Da but rather would appear at 66,420 Da in the resulting deconvolved mass spectrum. Thus, each peak will be rounded to the nearest 10 Da in this example. A sample rate of 1 Da would be needed to read a mass peak at 66,417 Da. Note: setting a smaller sample rate will slow down the algorithm in UniDec but will improve the precision.
Once the charge range, mass range, and sample rate have been set, click Run UniDec to perform the deconvolution. Clicking the Run UniDec button will create 3 new additional plots. The first, below the m/z spectrum, is a 2-D plot with charge on the y-axis and m/z on the x-axis. The charge vs m/z plot shows the charge states for each m/z value in the processed spectrum. You should see a general downward “swooping” trend of lower charge states at higher m/z. Below the charge vs m/z plot, the second plot is another 2-D plot with charge on the y-axis and mass, in Da or kDa (UniDec will automatically select the units depending on the mass range), on the x-axis. This plot shows the charge states for the deconvolved mass values. A single analyte should have multiple charge states at the same mass resulting in a vertical distribution on the plot. Finally, the third plot, to the right of the raw mass spectrum, is the zero-charge mass spectrum. The zero-charge mass spectrum is the deconvolved mass spectrum with intensity on the y-axis and mass, in Da or kDa, on the x-axis. It is the sum of the 2-D plot of charge vs. mass along the charge axis. Similarly, the sum of the 2-D plot of charge vs. m/z along the charge axis is shown in red on the top left figure to show the fit to the data.