Glycosylation is one of the most common and biologically important classes of protein modifications, yet it is also one of the most difficult to study. Monosaccharide composition is often discernible directly from accurate molecular mass (MS1); however, topology (monosaccharide connectivity) requires MS2, and glycosidic linkage information and anomericity may require MS3 if not combined with orthogonal insight. Byos® incorporates fragment analysis which reports on disambiguation of topological isomers directly from tandem MS spectra.
Released glycan workflows within Byos generate chromatogram mapping projects for the analysis of complex biomolecule samples. The processing steps include integration of traces across various detector types, peak and feature finding, and peak matching to biomolecule candidates. With the addition of tandem MS features, including the MS2 Data table and MS2 Plot widgets, users can use fragmentation data to better assist in the assignment of biomolecules to elution peaks. For the case of released glycans, the user can utilize these features to view fragment-matched and scored spectra to explore different glycan fine structures.
Fragmentation at the glycosidic bond between monosaccharides produces B, C, Y, and Z-type fragments. Cross-ring fragmentation, producing A and X-type fragments, may also occur, from which glycan linkage can be determined.
In order to utilize MS2 fragmentation options, users must enter the following advanced commands into the workflow:
[Byomap]
EnableMS2=true
Ms2Tolerance = 0.1 da
Bonus tip: Fragment annotations work for both DDA and most DIA data.
To learn more about how to use the Released Glycan workflows, users can refer to our workflow guide , advanced tips, and cartoon customization articles on our knowledge base. Additional case studies of MS2 discerned release glycans can be found on proteinmetrics.com > Resources > ASMS 2023 poster titled "Automated Spectrum Annotation and Structure Disambiguation of Released N-linked Glycans".