The Williamson Lab

Envelope

Documentation

0) The File Menu

The file menu can be used to open data files (also accomplished by clicking the "+" button while the "Data" type is selected) as well as to export graphic files in PDF or PNG format. Selecting "Export Calculated Spectra" exports all spectra of the type "Calc" or "Sum" as a comma-separated-values text file (.csv).

1) The Main Display Area

This is the area where both data and calculated spectra are displayed. M/z is displayed along the x-axis, and intensity along the y-axis. You can interact with this area in the following ways:

Click and drag to select an area and zoom in on it.
Ctrl-Click/Right-Click and drag to move the spectrum around in the area.
Double-Click to reset the Autorange feature, and zoom out to show all of the spectra

2) Controls Directly Below the Main Display Area

The "+" and "-" buttons are used to add or remove spectra of the specified type (Calc or Data). If "Calc" is selected, the newly added spectrum will default to atom-based labeling, but with natural abundance values. If "Data" is selected a dialog box will open prompting you to select a data file.

Here you can specify either protein or RNA, the sequence of the peptide or oligonucleotide (including modifications), and the charge state. The charge defaults to positive for proteins and negative for RNA.

The Fraction here displays the value defined in the listbox listing all the spectra (3). It is calculated based on the amplitudes of the spectra included in the definition.

In order to calculate the Fourier Transforms necessary for the isotope distributions, Envelope calculates data for a discrete number of points (which must be a power of 2), at a density of 100 points/dalton. The total range calculated is the number of points divided by 100, then divided by the charge of the peptide. By default, Envelope calculates 2¹³ points (8192), which translates into about 82 daltons/z range for a +1 peptide or 41 daltons/z range for a +2 peptide. Large, labeled peptides may exceed the available range, causing the spectrum to wrap around - in this case, increase the calculated range above default. The calculated range can also be decreased for speed advantages when dealing with small peptides.

3) The Spectral Listbox

The Spectral Listbox contains information about all of the different spectra, both experimental data and calculated, which are currently active in the program.

•	(Calc only) This checkbox allows you to include or exclude the designated spectrum from the calculations. Useful for rapidly switching between different labeling patterns.
Name	This is simply a user-defined name for the different spectra. It defaults to the filename for experimental data, and is included in the .csv output of calculated spectra.
Type	This indicates the type of spectrum (Data, Calc or Sum)
Show	These checkboxes indicate whether or not to display the given spectra in the main window. "Calc" spectra which are not displayed are still included in the calculations and thus the "Sum" spectrum (unless they are excluded via the • column)
Amplitude	(Calc only - 2 columns) The amplitude is the factor by which the unit spectrum is multiplied to give the final spectrum. This can be adjusted higher or lower to fit experimental data.
Chi Squared	(Data only) The Chi Squared is a goodness of fit figure automatically calculated for each data file compared to the "Sum" spectrum.
X and Y	(Calc only) Use these checkboxes to define the Fraction (the result of which is displayed in section 2, see above) in terms of the amplitudes of the different species.

4) & 5) Controls to Define Labeling Patterns

The controls in (4) and (5) reflect the spectrum which is currently selected in the listbox (3), and apply only to "Calc" type of spectra. The drop-down menu allows you to select between three different labeling styles:

None	Specify this for unlabeled species. This is effectively the same as specifying "Atoms" or "Residues", and leaving the natural abundance values specified.
Atoms	Specify this for atom-based labeling patterns (ie uniform labeling with 50% ¹⁵N)
Residues	Specify this for labeling with specific residues (ie ¹³C Leucine)

The "Defaults" button will replace any specified labeling parameters you have for either atom-based or residue-based labeling with natural abundance values.

When "Atoms" is selected as the type of labeling pattern, the "Atoms" tab becomes available in the labeling pattern definition area. You can directly specify the ¹³C, ²H and ¹⁵N content of the peptide as a fraction from 0 to 1. Displayed are values for natural abundance ¹³C and ²H content, and labeled with 50% ¹⁵N.

When "Residues" is selected as the type of labeling pattern, each of the individual amino acid and nucleotide tabs become available in the labeling pattern definition area. You can directly specify the ¹³C, ²H and ¹⁵N content of each residue as a fraction from 0 to 1. Furthermore, you can specify the actual fraction of the particular type that is labeled, in the "Fraction" field. Shown above is a labeling definition for Alanine. The Fraction is set to 0.5, meaning 50% of the Alanines in the species are labeled. The ¹³C value is set to 0.97, indicating that those Alanines which are labeled have a ¹³C content of 97%.

To accomodate hydrogen exchange, it is possible to define a more detailed labeling pattern for the hydrogen atoms of a residue. There are two different types of Hydrogens you can define, H2A and H2B. Each of these can have its ²H content defined individually. By default H2A will account for all of the hydrogen atoms in a residue, and H2B none of them. By using the drop-down menus you can change the assignemnt to any combination you wish. Consider the above example using Leucine. Leucine has 11 hydrogen atoms in total. H2A is defined to include 9 of those, labeled with 97% ²H. H2B is defined to include just 1 hydrogen, labeled with 85% ²H. All hydrogens not included in H2A or H2B (in this case the single remaining hydrogen) are assumed to have natural abundance ²H content.

6) Global Spectral Properties

Baseline	A baseline correction factor applied to all calculated spectra.
Gaussian Width	This determines the peak width of peaks in the calculated spectra. This is not the peak width itself, but rather a variable in a gaussian function. Larger values actually result in narrower peaks.
Offset	This defines a mass offset correction which is applied to all calculated spectra, in order to accomodate a machine-based offset when fitting to experimental data.

7) Appearance of Traces

Here you can define the visual appearance of both calculated spectra and experimental data. Choose between displaying lines and/or points, the color of the trace and the line width. For experimental data only there is the option to color points based on the goodness of fit to the Sum of calculated spectra. This gives a visual indication of where the worst fit areas are.

8) Miscellaneous Options

Enabling this option will result in a grid being drawn in the main display area.

The Set Autorange button will optimize the main display area to display all of the spectral content.

The Go button initiates calculation of spectra. When the Live option is enabled, spectra will automatically be calculated and redisplayed in response to changes to the various labeling parameters.