Post translational modifications (PTMs) influence the biochemical and therapeutic properties of proteins and can occur either intracellularly or during biopharmaceutical manufacture and storage. Identifying and quantifying PTMs, such as deamidation, oxidation, and glycosylation, is important to determine the stability, structure, and efficacy of therapeutic proteins.
Byos provides several different label-free quantification ways to calculate the % modification within the Peptide workflows. The default reports will automatically perform these calculations.
XIC Ratio% in the Inspection View
The XIC Ratio % column provided in the inspection view is calculated per modification and per charge state, as shown in eq 1:
%Mod by ModName Across Samples in the Report (2nd Tab)
The PTM default report’s 2nd tab uses the same XIC Ratio % calculation as shown in eq 1 and then arithmetically averages across charge states. Quantification based on the simple XIC Ratio % is often sufficient and is accurate when the extent of modification is low.
The table in the PTM default 2nd tab is organized by each modification type (Mod. Names).
%Mod (Multi Mods per Peptide) in the Report (4th Tab)
When there is significant degradation or modification it may be desirable to ratio against a sum of all relevant modifications. This will both account for a loss of wildtype peptide and account for the presence of significant amounts of other modified forms. The PTM default report’s 4th tab does not utilize "XIC Ratio%" values. Rather, it takes into account all other modifications of the same sequence peptides, and then re-calculates relative abundances via normalization on the peptide level. 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 using the XIC area summed IsoX normalized dynamic column. This table also sums XICs of different charge states before calculating ratios. The calculation can be considered as a weighted average as shown in eq 2:
The table in the PTM default 4th tab is organized by peptide sequence (Sequence (unformatted)).
Note: For more information on these calculations, please refer to the Knowledge Base article Asparagine Deamidation Quantified (link).
The wildtype peptides are hidden in the table by default. To show the wildtype peptides first select Show configuration in the top left, then select the down arrow in the Mod. Name field box, check the wildtypes box, and select Apply.
All Peptide PTM Quant in the Report (Tab Preset)
The All Peptide PTM Quant tab allows the user to group peptides based on a specific modification location or variable position. This will take into consideration all modified and corresponding wildtype peptides with a common variable position regardless of digestion specificity. The All Peptide PTM Quant tab will use the same calculation as the PTM default report’s 4th tab (eq 2). This tab, by default, will use XIC AUC values for % modification calculations; however, the user can change this field to XIC area summed IsoX normalized if desired.
The table in the All Peptide PTM Quant tab is organized by variable position (Var. Pos. Protein).
To enable this tab in the report, select File>Presets>Tab presets> Blgc_Pivot_All_Peptide_PTM_Quant.tabc
The All Peptide PTM Quant tab will also hide the wildtype peptides by default. To show the wildtype peptides first select Show configuration in the top left, then select the down arrow in the Mod. Name field box, check the WT DUPLICATE box, and select Apply. This tab uses a WT DUPLICATE script to group WT peptides and quantify modifications using all relevant peptides.