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.

中文翻译：

---

相似但不相同：人血清，牛血清和重组人胎球蛋白的蛋白形式的深度分析。

胎球蛋白，也称为α-2-HeremansSchmid糖蛋白（AHSG），属于分泌到血液中的一些最丰富的糖蛋白。在血液中，胎球蛋白展示出作为金属和小分子的载体的功能。牛胎球蛋白具有3个N-糖基化位点和建议的六个O-糖基化位点，经常被用作模型糖蛋白，以测试糖蛋白组学中的新型分析工作流程。在这里，我们使用三种不同生物学来源的蛋白质（人血清，牛血清和重组人胎蛋白）在HEK-293细胞中表达，深入表征和比较胎球蛋白，目的是阐明这些蛋白质与翻译后蛋白之间的异同他们所拥有的修改。将高分辨率的天然质谱数据和以糖肽为中心的LC-MS分析数据相结合，我们定性和定量地收集有关胎球蛋白蛋白质成熟，N-糖基化，O-糖基化和磷酸化的信息。我们提供直接的实验证据，即人血清和部分重组蛋白都被加工成通过单个链间二硫键连接的两条链（A和B），而牛胎球蛋白仍然是单链蛋白。尽管从牛到人保守了两个N-糖基化位点，一个O-糖基化位点和一个磷酸化位点，但是修饰的化学计量和它们所具有的特定糖型是非常不同的。比较血清和重组人胎球蛋白，我们观察到血清蛋白具有简单得多的蛋白形式，表明重组蛋白不是理想的工程设计来模仿人血清胎蛋白。