Summary:
- Byos isotope selection for XIC AUC measurement
- Definition of the Byos iso field
- Explanation of the {XIC area summed isoX normalized} pre pivot script
Isotope Selection for XIC AUC:
Byos XIC AUC is measured by integrating the area under the curve of an extracted ion chromatogram, plotted using a single isotopic peak, which is selected based on the MS extract options Isotope table and the Calc. M of the peptide. The MS extract options m/z integration window (ppm) defines a ppm width for the XIC integration. By default, Byos peptide workflows (PTM, HCP, SS, SVA, etc.) use the Isotope table shown in Figure 1, set to select the most abundant isotope peak based on an averagine model, and a m/z integration window (ppm) setting of 18.00.
Figure 1: The MS extract options and Isotope table in the Processing nodes section of the workflow.
The Byos iso field indicates which isotope peak was used to determine XIC AUC. The project investigation Isotope Plot highlights the isotope peak used to determine XIC AUC with a pink transparent bar as shown in Figure 2 below. The width of the pink transparent bar is equal to the MS extract options m/z integration window (ppm) setting.
Figure 2: The Isotope Plot for a peptide. The blue text is added to show what different iso values mean. The iso=0 or monoisotopic peak is used for determining the XIC AUC value for this example peptide, as indicated by the pink transparent bar.
NOTE: With the exception of deamidation modified peptides (discussed below), all other modified peptides with a corresponding wildtype peptide always use the same isotopic peak for XIC AUC as their wildtype counterpart, regardless of the measured Calc. M of the modified peptide.
{XIC area summed isoX normalized}:
When Deamidation Quantification is enabled (enabled by default for Byos PTM) deamidation modified peptides always use the monoisotopic peak (iso=0) for XIC AUC. The corresponding wildtype peptide iso value is determined based on the Calc. M and MS extract Isotope Table as described in the section above. This means deamidation and corresponding wildtype XIC AUC are sometimes determined using different isotope peaks. See the linked knowledgebase article Asparagine Deamidation Quantified for more explanation of Deamidation quantification in Byos.
{XIC area summed isoX normalized} is a pre pivot dynamic column script that corrects XIC AUC for deamidated peptides when the isotope peak (or iso) used for XIC AUC determination differs from the corresponding wildtype. Deamidated peptide {XIC area summed isoX normalized} is calculated using the deamidated peptide XIC Ratio% and the wildtype XIC AUC with the equation below.
NOTE: For all wildtype peptides and most modified peptides, {XIC area summed isoX normalized} is equal to XIC AUC.
VVSVLTVLHQDwLnGK iso selection and {XIC area summed isoX normalized} calculation example:
The table in Figure 3 shows Calc. M, iso, XIC AUC, {XIC area summed isoX normalized}, and XIC Ratio% values for wildtype, deamidated, and oxidized versions of the VVSVLTVLHQDwLnGK peptide sequence.
Figure 3: A report tab showing key values for calculating {XIC area summed isoX normalized} for a deamidated peptide with red boxes highlighting values used for the example calculation shown below. The green box shows the iso values for each peptide signal.
The wildtype isotope peak is selected based on the MS extract options Isotope table (Figure 1) and Calc. M. The Calc. M of the VVSVLTVLHQDWLNGK wildtype peptide is 1807.00 Da and falls between the MS extract options Isotope Table range of 1800-3000 Da, meaning the +1 isotope peak (iso=1) is used for XIC AUC determination. The Dioxidation modified peptide uses iso=1, aligned with its corresponding wildtype peptide. Because Deamidation Quantification is enabled, the deamidated peptide uses iso=0.
For the wildtype and Dioxidation modified peptide signals, {XIC area summed isoX normalized} is equivalent to XIC AUC because XIC AUC is measured using the wildtype isotope peak (iso=1). For the deamidated peptide signals, {XIC area summed isoX normalized} is XIC AUC normalized to account for the differing isotope peaks used between deamidation and wildtype signals. An example of the normalization calculation using Deamidation N>D z=2 peptide signal (see red boxes in Figure 3) is shown below.
Example {XIC area summed isoX normalized} calculation:
{XIC area summed isoX normalized} = (WT XIC AUC)*(Deamid. XIC Ratio%)/(100 - Deamid. XIC Ratio%)
= (207,938,000)*(0.50950517)/(100 - 0.50950517)
= (207,938,000)*(0.50950517)/(99.49049483)
= 1,064,880