5. HDXplotR

Purpose

The HDXplotR tab is used to plot the centroids as a function of time, convert said centroids to number of unexchanged sites NUS using the deuterium content, charge, and reference centroid m/z, and plot the results. Non-linear fitting of the results can also be achieved.

Principle

Centroid masses $(m/z)_t$ are extracted from the selected spectra by calculating the average isotopic mass $m_i$ weighted by the abundance $a_i$:
$$(m/z)_t = \frac {\sum_i{m_ia_i}}{\sum_i{a_i}}$$

The NUS is defined as the measured mass shift between the sample centroid mass $(m/z)_t$ at a time t calculated above and that of the fully-exchanged reference centroid $(m/z)_∞$ (user supplied, can be determined in OligoRef), converted to mass units by multiplying by the charge |z| (user-supplied), and divided by the apparent isotopic mass shift for a single site $m_s$:
$$\ NUS_t = \frac {[(m/z)_t-(m/z)_∞]}{\Delta m_s}|z|$$ with $$\Delta m_s=(D_0-D_∞)(m_D-m_H)$$ $D_t$ is the molar fraction of deuterium at a time $t$ and $m_H$ and $m_D$ the molecular mass of hydrogen and deuterium respectively

Input

In the sidebar, the user can input:

1. The choice of timescale (manual or TIC)
2. The deuterium content pre- and post-exchange
3. The m/z of the fully-exchanged reference
4. The charge of the analyte (the reference and the kinetics data must have the same charge)
5. Non-linear fitting parameter initial values, for a double exponential decay

In the main window, the user can select the data points to be converted to NUS by clicking on the corresponding table lines, or simply tick the select all box, from the data table (see output).

Figure theming

See the figure customization and export page.

Output

The output is composed of four plots and two tables:

• The raw exchange plot displays the centroid values as a function of time
  • The time axis depends on the selected timescale (manual or TIC)
  • All species are displayed in the same color (gray by default)
• The NUS exchange plot, which is the same as the raw plot, except for the y-axis that is now NUS values
  • Species are colored differently
  • Non-linear fitting is also displayed there
• The deconvoluted exchange plot
  • NUS as a function of time for the populations of high and low exchanges determined in [MSstackR]
• The deconvoluted population abundances
  • Displays the relative contribution of each population normalized between 0 and 1
• The data table that presents the centroid and NUS values for each data point, alongside the time (for all timescales), the species number, the reference centroid and charge used in the NUS calculation, and the selected timescale (hidden by default)
  • The user-selected lines are colored in orange
  • The table is sortable, searchable and exportable in the clipboard or csv/Excel files