Isobaric Aspartic Acid (Asp, D) and iso-Aspartic Acid (isoAsp, isoD) residues are indistinguishable in MS/MS spectra using CID or HCD fragmentation, though ETD may result in signature ions z-57 and c+57, which are automatically annotated by Byonic and Byos. Normally, a peptide with D and isoD variants appears as a single wildtype (unmodified) peptide. To calculate the relative amount of isomerization, it is necessary to separate the peaks using the retention time difference between the isomers.
There are manual, semi-automated, and fully-automated methods to calculate the relative populations (%PTM) of each isomer. All of these methods involve creating a separate peptide for the isoD variant, labeling it, and generating relative population % in the report.
Below, we cover isomerization in native Asp residues (D→iso-D). For isomerization related to Asn deamidation (N→D or N→iso-D), please see the application note titled “PTM Analysis: Asparagine Deamidation Quantified by Byologic”, accessible on our website.
Byos (legacy Byologic) detects multiple elution peaks in a peptide’s XIC by default. These are called XIC proposals. They can be seen in the Peptides table under XIC proposals column, and visualized in the XIC plot as follows:
The user can right click on the peptide in Peptides table, and click Apply proposed XIC splits (with charge states combined). This will create a new in-silico peptide, and automatically set the XIC integration limits to the XIC proposals. In order to distinguish the D and isoD variant, add a Label to the newly created peptide. Eg. “isoD”.
When a wildtype peptide exhibits multiple elution windows and contains a pre-defined motif (eg. DG, DS), Byologic can automatically split the peptide and label the variant as “isoD”. The automatic splitting and labeling requires advanced commands to be entered at project creation.
Although not automated, the manual method is the most straightforward to implement. It simply involves more clicks: Right click on the wildtype peptide and click on Create new peptide from current… A new peptide will be created. Note that the new peptide’s “in silico” column will show “Yes”, and the Mod. Summary column is still empty, because Byologic simply copied a wildtype peptide. To distinguish the new peptide add a Label to this peptide. Here we labeled it “isoD”.
Change XIC interval to the isoD peak only. Similarly, exclude the isoD peak from the parent peptide’s XIC interval. The relative abundance of isoD will be automatically shown in the report tables.
In all of the above methods, the relative abundance of isoD variant is calculated and
shown in the report. The default (PTM) report’s fourth tab "% Mod (Multi Mods per
Peptide)" does this automatically, however, wildtype peptides need to be shown, which
are hidden by default. See instructions in the screenshot below.
In the below Table, relative abundance of D and isoD variants of peptide FNWYVDGVEVHNAK is shown in four samples. Percentage values are calculated by comparing integrated XIC values: XIC_isoD / (XIC_D + XIC_isoD). The below data shows a clear increasing isoD abundance in stressed samples compared to the