Protein Metrics software includes a powerful reporting feature which outputs a project summary, various types of tabular data, charts, and plot images. Some basic calculations are readily available, such as normalization, summation, etc. Often, users may need a custom calculation or a specific text label to represent their results in a different way than provided by default. The reports include a feature called “dynamic columns” to provide a flexible option to build these custom columns.
A number of commonly used dynamic columns are provided with the software. These can be added to the Report by choosing them in the
File Menu→Presets→Pre pivot columns. For descriptions of these, or to learn how you can create your own dynamic columns, please contact us.
One dynamic column in particular is used in the calculations in the default PTM workflow report, which brings us to the next topic:
What does “XIC Area Summed isoX Normalized” mean?
When calculating XIC Ratio% value of deamidated peptides, Byos compares the monoisotopic ion XICs of both deamidated and corresponding wildtype peptides. The rationale behind this can be found in our application note titled “PTM Analysis_N-Deamidation Quant”. The XIC Ratio% column, shown in the inspection view, is calculated per modification and per charge state, simply as
XICmod÷ (XICmod+ XICwildtypes).The PTM default report’s 2ndtab uses this value, arithmetically averaged across charge states and possibly missed cleavages.
The report's last tab, however, does not utilize "XIC Ratio%" values. It takes into account all other modifications of the same sequence peptides, and re-calculates relative abundances via normalization. If one of the modifications is deamidation, the XIC of the deamidated peptide is normalized, so that the isotopes used to generate XICs match between deamidated, wildtype, and other modifications. This table also sums XICs of different charge states before calculating ratios. The calculation can be considered as weighted average:
“XIC Area Summed isoX Normalized” is the dynamic column which performs the normalization. It normalizes XIC area summed values for peptides that contain deamidation alongside other modifications, because the XIC area summed may be calculated using different isotopes for the deamidated peptide (monoisotope) and the other modifications (determined by MS extract options table). This column normalizes deamidated peptide XICs using the formula:
Wildtype XIC area summed * XIC Ratio/ (1- XIC Ratio).