TY - JOUR
T1 - Similar albeit not the same; in-depth analysis of proteoforms of human serum, bovine serum and recombinant human fetuin
AU - Lin, Yu-Hsien
AU - Franc, Vojtech
AU - Heck, Albert J R
PY - 2018/7/2
Y1 - 2018/7/2
N2 - 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 a dozen O-glycosylation sites, has been often used 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 recombinant proteins are processed into two chains (A and B) connected by a single inter-chain 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-structure of these two proteins are alike, they represent quite distinct proteins when their glycoproteoform profile is also taken into consideration.
AB - 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 a dozen O-glycosylation sites, has been often used 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 recombinant proteins are processed into two chains (A and B) connected by a single inter-chain 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-structure of these two proteins are alike, they represent quite distinct proteins when their glycoproteoform profile is also taken into consideration.
U2 - 10.1021/acs.jproteome.8b00318
DO - 10.1021/acs.jproteome.8b00318
M3 - Article
C2 - 29966421
SN - 1535-3893
VL - 17
SP - 2861
EP - 2869
JO - Journal of Proteome Research
JF - Journal of Proteome Research
IS - 8
ER -