Tutorial 1: Confirming Polyisotopic Compounds in a Mass Spectrum
In this tutorial we will use IsoGlyph to confirm if a polyisotopic compound is present in a noisy experimental mass spectrum or not.
Preparing the data
Section titled “Preparing the data”The mass spectrum used in this tutorial can be downloaded here. It is a medium resolution spectrum that was produced by detecting a low concentration of HgCl2 with iodide reagent ion. The ion detectable in the spectrum is HgI3- and the signal-to-noise ratio is low.
The file HgI3_low_signal.tsv contains two tab-separated columns without a header. The first column corresponds
to m/z and the second column - to peak intensity. This spectrum has already been centroided. IsoGlyph also supports
profile spectra, which are supplied in the same tab-separated format, but the second column corresponds to raw signal
in that case. The easiest way to generate a .tsv file from your existing spectrum is to open it in Excel, copy
m/z and intensity columns, and paste them into a blank text file.
100.0025 6.183e0100.0349 2.436e1...Getting started with IsoGlyph
Section titled “Getting started with IsoGlyph”If you haven’t already, download the latest version of IsoGlyph for your platform. Extract the executable and move it to a convenient location. Open the program. Populate each field as directed below. Brief explanations of what each field does are provided here. For more detailed explanations of each input, click on the links in the list.
| Input | Description | Value |
|---|---|---|
| Path to spectrum | Absolute or relative path to the .tsv spectrum that you downloaded |
|
| Peak separation | Smallest allowed peak separation in the theoretical spectrum (amu) | 0.02 |
| Min. rel. intensity (theoretical) | Minimum allowed peak intensity in the theoretical spectrum relative to the tallest peak | 0.001 |
| Min. abs. intensity (experimental) | Minimum allowed peak intensity in the experimental spectrum | 0.01 |
| Tolerance | Maximum allowed m/z drift of peaks within a mass spectrum relative to each other (amu) | 0.1 |
| Find N candidates | Number of matched candidates to plot and display | 1 |
| Estimate and subtract background | Optionally, estimate and subtract local background noise near the matched spectrum | on |
| Search window width (tab) | Potential matches will only be searched for in the vicinity of the theoretical spectrum | |
| Width | Width of the search window, centered at the theoretical mass spectrum (amu) | 10 |
| Centroid spectrum (tab) | Spectrum is already centroided and each point will be treated as a peak | |
| Search mixture (tab) | Search will be performed either for a single compound or a mixture with known composition | |
| Compound | Chemical formula of the compound that will be searched | HgI3 |
| Fraction | Fraction of the compound in a mixture | 1 |
| Charge | Charge of the compound | 1 |
| Experimental plot scale | Switch between linear and log intensity scale on the plot with the full experimental spectrum | log |
Running IsoGlyph and interpreting the results
Section titled “Running IsoGlyph and interpreting the results”With the example spectrum HgI3_low_signal.tsv downloaded and all IsoGlyph parameters set to values given in the
table above, click on “Run search” in the bottom left corner. The search should only take a few seconds and at the end,
some plots and text output will be displayed: