MineXpert3 User Manual
- Preface
- 1 Generalities
- 2 Configuring the application preferences
- 3 The program's graphical user interface
- 4 Mass Data Integrations Featured by MineXpert
- 5 Mass Spectral Deconvolutions
- 6 Isotopic cluster calculations
- 7 The processing flow in a data exploration session
- 8 Recording data exploration discoveries
- 9 Scripting Interface
- A GNU General Public License version 3
5 Mass Spectral Deconvolutions
When analysing a mass spectrum, two major deconvolutions are performed to get back to the Mr mass of the analyte while reading m/z values: the charge-based deconvolution and the monoisotopic cluster-based deconvolution. In the following sections, both deconvolutions are described.
Before delving into the deconvolutions, it is necessary to present two menu options that are found in the plot widgets contained in the Mass spectra window: the menu items under the Deconvolutions menu (Figure 5.1, “Mass spectrum plot widget-specific deconvolution menu” ).
The two menu items are needed to configure the mouse-based deconvolution of mass spectra.
Figure 5.1: Mass spectrum plot widget-specific deconvolution menu #
These two menus allow one to set parameters for the deconvolution (see text for details).
5.1 Deconvolution based on charge state #
In this kind of deconvolution, at the present time, the software assumes that the ionization agent is the proton and that the ionization is positive.
The deconvolution is based on the determination of the distance
between two peaks —consecutive or not— of a given charge
state envelope. When the user 
-click-drags the cursor from one
peak to another, the program tries to calculate if the distance between
two peaks matches one or more charge difference(s). If so, it computes
the molecular (Mr [12C]-relative molecular mass) mass of the analyte.
Note: The mouse drag movement is significant
The Mr value that is computed is for the analyte below the mass peak at which the mouse drag moment operation started.
Top panel of Figure 5.2, “Charge state-based mass deconvolution” shows the charge state envelope-based deconvolution process for a protein of Mr≍8566 Da. In the top panel, the deconvolution has involved two consecutive peaks ( span> is 1). The mouse drag movement occurred from left to right. Thus, the m/z value chosen for the computation is that under the left peak (start point of the movement). The status line at the bottom of the plot indicates the selection range, the delta movement on the x-axis, the computed charge, the Mr value chosen for the calculation (the Mr value at the start of drag movement) and finally the calculated Mr value.
This “two consecutive mass peaks” method is the default method. However, it might happen that no clearly visible mass peak is available around one nice peak that might be chosen as the start of the mouse drag operation. In this case it is possible to define a different span> between two peaks elected for the deconvolution (see Figure 5.1, “Mass spectrum plot widget-specific deconvolution menu” ). In the figure, than span has been set to 2, which means that the mouse drag movement encompasses three mass peaks: the movement start peak, one peak in the middle and finally the movement stop peak (the span is thus of two intervals between the extreme peaks).
The bottom panel of the figure now displays the same Mr value for the protein even if the span is now of two intervals.
Deconvolution approach using two peaks belonging to the same charge state envelope. The top deconvolution involves two consecutive mass peaks (peak span value is 1). The bottom deconvolution involves two non-consecutive peaks (peak span value is 2). The Mr value, expectedly, did not change whatever the configured span.
Figure 5.2: Charge state-based mass deconvolution #
Note
The charge calculation, which is at the heart of the deconvolution, almost never produces an integer value with no fractional part (say, charge z=15.0) because it is almost impossible to drag the mouse cursor the exact number of pixels that would match a m/z range leading to such an integral charge value. Almost always, the charge that is calculated looks like 14.995 or 15.001, for example. This is due to the fact that the mouse moves at discrete positions on the screen and these positions might be more or less far apart, depending on the mouse capabilities and on the current zoom factor over the mass spectrum region of interest.
It is advised to zoom-in as much as possible over the peaks at hand so as to minimize the difficulties above. It may happen, however, that even zoomed-in peaks are not sufficiently distant to allow a charge calculation. In this case, reduce the stringency over the fractional part that is allowed in the charge (see menu item Set charge minimal fractional part at Figure 5.1, “Mass spectrum plot widget-specific deconvolution menu” ). By default, the stringency is set at 0.99, that is, any calculated value that has a fractional part either superior or equal to 0.99 or inferior or equal to 0.01 would lead to a successful round-up/round-down to the nearest integer value. Outside of the [0.01-0.99] interval, no charge calculation is performed and thus no deconvolution is performed. When the stringency is too high, reducing it will allow the deconvolution to be carried-over. General experience is that setting that value to 0.997 is fine for most situations and provides very reliable results.
5.2 Deconvolution based on isotopic cluster peaks #
In this kind of deconvolution, the user -click-drags the cursor
between the first two peaks (when possible) of the isotopic cluster. The
charge state of the ion is the inverse of the distance between the
two consecutive peaks (that is, the m/z delta value). Figure 5.3, “Isotopic cluster-based mass deconvolution”
shows that deconvolution process at work.
Top panel: deconvolution involving a left-to-right mouse drag movement. Bottom panel: deconvolution involving a right-to-left mouse drag movement. The calculated Mr values differ.
Figure 5.3: Isotopic cluster-based mass deconvolution #
Note: The mouse drag movement start position is significant
The -click-dragging direction (left→right or
right→left) determines the final Mr that is computed because
that value is calculated for the peak under the mouse when the mouse
drag movement is initiated. This is visible in the two panels of Figure 5.3, “Isotopic cluster-based mass deconvolution” , where the
top panel shows the Mr computed for the left peak and the bottom
panel shows the Mr computed for the right peak. Since the ion is
monocharged, the difference is 1 Da.
This is a significant departure from the previous versions, where the postulate was that the single real peak of interest in an isotopic cluster was the left-most monoisotopic peak. Since this software has been used by scientists in research projects using almost 100 % labelled bacteria (with [13C] and [15N]), that concept has become moot. Indeed, analytes from these bacteria have their monoisotopic peak at the far right end of the isotoopic cluster.
The new behaviour allows the scientists to compute the Mr value of the peak of interest in an isotopic cluster, be that for a non-labelled or for a labelled analyte. See the following articles as examples of heavy isotope almost full labelling of bacteria.
Heavy isotope labeling and mass spectrometry reveal unexpected remodeling of bacterial cell wall expansion in response to drugs. Atze H, Liang Y, Hugonnet JE, Gutierrez A, Rusconi F, Arthur M. Elife, 2022, doi: 10.7554/eLife.72863, PMID: 35678393.
Peptidoglycan-tethered and free forms of the Braun lipoprotein are in dynamic equilibrium in Escherichia coli. Liang Y, Hugonnet JE, Rusconi F, Arthur M. Elife, 2024, doi: 10.7554/eLife.91598, PMID:39360705.
(p)ppGpp modifies RNAP function to confer β-lactam resistance in a peptidoglycan-independent manner. Voedts H, Anoyatis-Pelé C, Langella O, Rusconi F, Hugonnet JE, Arthur M. Nat Microbiol, 2024, PMID:38443580.
5.3 Reading the resolving power based on mass spectral data #
When -click-dragging the mouse cursor between two mass spectrum
locations of interest, the program computes the apparent resolving
power. This process is shown on Figure 5.4, “Calculation of the resolving power”
, where
the resolving power is calculated by dragging the mouse cursor from
one edge of a peak to the other at half maximum height (this is
called full width at half maximum [FWHM]
resolution).
Click-dragging the mouse cursor will trigger the calculation of the resolving power of the instrument. That value is printed in the status bar.