N-linked Glycosite Profiling and Use of Skyline as a Platform for Characterization and Relative Quantification of Glycans in Differentiating Xylem of Populus trichocarpa

Loziuk, P.L.; Hecht, E.S.; Muddiman, D.C.

Given the importance of glycomic characterization in biological systems, Muddiman DC and co-workers developed a new workflow for the combined profiling of deamidated glycosites and N-linked glycans in the differentiating xylem of Populus trichocarpa. The workflow involved a FANGS-INLIGHT coupled methodology, which featured hydrazide tagging (via the heavy/light INLIGHT reagent) of the enzymatically released N-linked glycans and bottom-up preparation of the deamidated glycoproteins from the filter, prior to LC-MS analysis. This protocol enabled 27 N-glycans and 502 glycosylated proteins to be identified, several of which have potential linkages to lignin biosynthesis. In addition, Skyline was demonstrated to accurately and precisely quantify N-glycans; thus, providing a new software solution for automated glycan analysis.