Anion Exchange Chromatography–Mass Spectrometry to Characterize Proteoforms of Alpha-1-Acid Glycoprotein during and after Pregnancy

Alpha-1-acid glycoprotein (AGP) is a heterogeneous glycoprotein
fulfilling key roles in many biological processes, including transport
of drugs and hormones and modulation of inflammatory and immune responses.
The glycoform profile of AGP is known to change depending on (patho)physiological
states such as inflammatory diseases or pregnancy. Besides complexity
originating from five N-glycosylation sites, the heterogeneity of
the AGP further expands to genetic variants. To allow in-depth characterization
of this intriguing protein, we developed a method using anion exchange
chromatography (AEX) coupled to mass spectrometry (MS) revealing the
presence of over 400 proteoforms differing in their glycosylation
or genetic variants. More precisely, we could determine that AGP mainly
consists of highly sialylated higher antennary structures with on
average 16 sialic acids and 0 or 1 fucose per protein. Interestingly,
a slightly higher level of fucosylation was observed for AGP1 variants
compared to that of AGP2. Proteoform assignment was supported by integrating
data from complementary MS-based approaches, including AEX–MS
of an exoglycosidase-treated sample and glycopeptide analysis after
tryptic digestion. The developed analytical method was applied to
characterize AGP from plasma of women during and after pregnancy,
revealing differences in glycosylation profiles, specifically in the
number of antennae, HexHexNAc units, and sialic acids.