Population-level analysis of glycoprotein glycoforms

The structure and function of many proteins are regulated post-translationally through glycan attachment. These glycans, assembled via competing enzymatic reactions, generate diverse glycoform populations – variants sharing a protein backbone but differing in glycan structures. While current analyses often focus on individual glycoforms, we demonstrate that population-level glycoform analysis – integrating spectral, biosynthetic, and physicochemical relationships – reveals new insights into glycoprotein regulation. Applied to immunoglobulin subclasses and antithrombin III (AT3), this approach provides comprehensive coverage of glycoform repertoires from human and murine plasma and biopharmaceuticals. It also enables sensitive quantification of glycosylation changes arising from in vitro manipulations or in vivo infections. Finally, we introduce a statistical framework adapted from ecological biodiversity studies, revealing that both IgG and AT3 exhibit skewed glycoform distributions shaped by biosynthetic constraints and degradation. Our findings demonstrate the added value of population-level glycoform analysis in understanding protein function and regulation through glycosylation.