Byos - Intact workflows assign and annotate neutral masses using the concepts of reference and delta masses. Reference masses are typically protein sequences or intact protein masses. If a sequence is provided, Byos calculates the average mass of the protein using its atomic composition and some optional settings such as if the protein N-term Q is cyclized to pyroQ, if the C-terminal Lysine is clipped, and if disulfide bonds are present or not. For reference, Byos uses average atomic masses based on isotope abundances in natural earth substances, which are documented in IUPAC technical report (Coplen TB et al. 2002 Pure Appl Chem. 74:1987). If the user needs to match the sequence-to-mass calculation to an existing SOP, the average atomic masses are configurable in the Building Blocks tab. A final hint about reference masses: the sequence can be modified by a mass. For example, one can enter “PROTEINSEQUENCE+215.3” in the sequence field to add 215.3 to the calculated average mass of the protein. This is useful in cases where there is a fixed modification in the protein with known mass but unknown elemental composition. If the elemental composition is known, the modification can be entered as a custom Letter in the Building Blocks tab.
Delta masses are the equivalent to variable modifications in Intact analyses. If an observed deconvoluted mass matches to any possible reference+delta mass combinations within the match tolerance, the observed mass will be automatically annotated. In Delta Masses table, a line with 0 mass must always be present to match unmodified reference proteins. All other delta masses are optional, can be checked, unchecked, and modified via double click. New delta masses can be added to the table using the “add row” button, or by importing from a CSV file, for which a template can be found in the program installation folder as mass_tables.csv.
As of version 3.7, adding combinatorial delta masses is also possible. For example, when performing ADC analyses, the user may want to add different drug loads and free-ended linker combinations to the list of delta masses. This is accomplished as shown in the following screenshots:
With just a few clicks, multiple drug loads and drug ± linker combinations are added as delta masses