Tutorial 5: Back exchange correction

The update to Deuteros 2.0 added a back exchange facility that allowed users to additionally import back exchange data for correcting peptide uptake levels.

Back exchange calculation requires an extra state in the cluster file that the user supplies to Deuteros. The 'control' state describes the minimally and maximally deuterated levels for peptides in the dataset. The steps for correction are described below:

Aggregating replicate peptide data into single values

As with any data imported into Deuteros, the first step in processing the cluster file is to aggregate the replicate-level data into single uptake values that represent a single peptide at a single exposure time. If multiple charge states are available for a set of replicates, the "best" charge state is first selected based on XX.

Calculate peptide back exchange

The next step involves calculating the back exchange level for each peptide in the control state to generate a look up table that can be used later on. We use the formula,

$bx = (1-\frac{m_{max}-m_{0}}{N\times D_{frac}})\times 100$

Where, $m_{max}$ is the mass of the maximally labelled peptide, $m_{0}$ is the mass of the minimally labelled peptide, $N$ is the max uptake of the peptide and $D_{frac}$ is the deuterium fraction [0-1] in the labelling buffer.

The values of $m_{max}$ and $m_{0}$ are calculated from the cluster file by multiplying the average replicate m/z (center) with the charge state (z) of the peptide.

Applying back exchange correction

To apply the correction to State A and State B peptides, we calculate each peptide's $m_{max}$ and $m_{0}$ in a similar fashion to above and then its corrected absolute uptake $DU^{\prime}$ as,

$DU^{\prime} = \frac{m - m_{0}}{m_{100} - m_{0}} \times N$

Example: Running the back exchange example file

1. Import the back_exchange_example.csv using the below settings and press import:

Note: although there are two dropdown menus for BX Correction, the same Control state should be set for both State A and State B.

A warning dialog box is displayed should any peptides in State A or State B not be found in the Control state, and thus cannot be corrected. Removed peptides can be found in the data table tab in the BX: Non-corrected table.

2. The first bit of analysis output after import is in the top-right summary box which shows the average and interquartile range of back exchange over the dataset:

Back-exchange (mean / IQR): 37.6% / 8.0%

3. Clicking on a peptide in the peptide tree will display the kinetics plot for the peptide. The legend in the top-left of the plot shows the back exchange percentage calculated for the peptide.

All subsequent analysis in the rest of Deuteros, e.g. coverage and global plots, will also use the corrected uptake values.

Data tables

Under the Data Tables tab, there are two data tables which are relevant to back exchange in Deuteros.

The BX: Control table shows a list of peptides from the control state as well as a final column that shows the back exchange percentage calculated for the peptide.

The BX: Non-corrected table contains peptides in State A and State B which were not found in the control state and thus cannot be corrected and have been removed.

The corrected uptake values replace those which are shown in the different State A (State), State B (State), States AB (Common), Δ(State B - State A) and Filtered Peptides tables.