Fetuin,
also known as alpha-2-Heremans Schmid glycoprotein (AHSG),
belongs to some of the most abundant glycoproteins secreted into the
bloodstream. In blood, fetuins exhibit functions as carriers of metals
and small molecules. Bovine fetuin, which harbors 3 N-glycosylation
sites and a suggested half dozen O-glycosylation sites, has been used
often as a model glycoprotein to test novel analytical workflows in
glycoproteomics. Here we characterize and compare fetuin in depth,
using protein from three different biological sources: human serum,
bovine serum, and recombinant human fetuin expressed in HEK-293 cells,
with the aim to elucidate similarities and differences between these
proteins and the post-translational modifications they harbor. Combining
data from high-resolution native mass spectrometry and glycopeptide
centric LC-MS analysis, we qualitatively and quantitatively gather
information on fetuin protein maturation, N-glycosylation, O-glycosylation,
and phosphorylation. We provide direct experimental evidence that
both the human serum and part of the recombinant proteins are processed
into two chains (A and B) connected by a single interchain disulfide
bridge, whereas bovine fetuin remains a single-chain protein. Although
two N-glycosylation sites, one O-glycosylation site, and a phosphorylation
site are conserved from bovine to human, the stoichiometry of the
modifications and the specific glycoforms they harbor are quite distinct.
Comparing serum and recombinant human fetuin, we observe that the
serum protein harbors a much simpler proteoform profile, indicating
that the recombinant protein is not ideally engineered to mimic human
serum fetuin. Comparing the proteoform profile and post-translational
modifications of human and bovine serum fetuin, we observe that, although
the gene structures of these two proteins are alike, they represent
quite distinct proteins when their glycoproteoform profile is also
taken into consideration
