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. 2018 Aug 3;17(8):2861-2869.
doi: 10.1021/acs.jproteome.8b00318. Epub 2018 Jul 13.

Similar Albeit Not the Same: In-Depth Analysis of Proteoforms of Human Serum, Bovine Serum, and Recombinant Human Fetuin

Yu-Hsien Lin  1   2 , Vojtech Franc  1   2 , Albert J R Heck  1   2
Affiliations

Similar Albeit Not the Same: In-Depth Analysis of Proteoforms of Human Serum, Bovine Serum, and Recombinant Human Fetuin

Yu-Hsien Lin et al. J Proteome Res. .

Abstract

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.

Keywords: N-glycosylation; O-glycosylation; alpha-2-HS glycoprotein; fetuin; glycopeptides; glycoprotein; hybrid mass spectrometry; native mass spectrometry; proteoforms; serum proteins.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Deconvoluted zero-charge mass spectra of (a) hFet, (b) bFet, and (c) rhFet. The zoom-ins on the right depict the most abundant peaks in the spectra to more clearly show the observed mass differences in each spectrum that originated mainly from distinct glycan moieties. The presence of a third N-glycosylation site in bFet increases the molecular weight and glycan heterogeneity of bFet compared to that of hFet. In (c), all proteoforms of rhFet are present in pairs, due to the co-occurrence of proteoforms with and without arginine, making the spectrum twice as complex as that of hFet. The glycan nomenclature used is indicated at the bottom.
Figure 2
Figure 2
Full native ESI-MS spectra of intact hFet sprayed from aqueous ammonium acetate. (a) Schematic cartoon showing that the B chain is connected to the A chain by a disulfide bridge. (b) Full native ESI-MS spectra of hFet upon treatment by DTT. The released B chain and A chain are observed. The peaks at m/z of 914.19 and 1132.93 correspond to the B chain and B chain with 1 O-glycan (HexNAc1Hex1Neu5Ac1), respectively. Comparing the most abundant peak on the charge state 13+ with m/z of 3380.24 in (a) and 3119.63 in (b), the mass difference indeed originates from the released B chain harboring the O-glycan. The dashed line box represents missing C-terminal A-chain arginine.
Figure 3
Figure 3
Overview of the observed qualitative and semiquantitative site-specific glycosylation in (a) hFet, (b) bFet, and (c) rhFet. The conserved glycosylation sites are depicted in the same column. Relative abundances of peptide proteoforms were estimated from their corresponding ion chromatograms (XICs). On a given modification site, the abundance of peptide proteoforms were normalized to 100%. All O-glycan structures and N-glycan structures attached to the 3 most abundant peptide isoforms of each site are depicted; further details of occupancy on each site is provided in the Table S1. (X means unmodified.)
Figure 4
Figure 4
Overall comparison of the protein structure and occurring PTMs on hFet, bFet, and rhFet. After (a) translation from single transcript, single-chain preproteins of hFet and bFet contain 367 and 365 amino acids, respectively. Then (b) N-glycosylation occurs in the endoplasmic reticulum followed by the modification of O-glycosylation sites. As in Figure 3, we depict the most abundant proteoforms at each glycosylation site. Distinctively, a high degree of fucosylation occurs on the N-glycosylation sites of rhFet, not observed for hFet. Also, bFet contains mainly triantennary complex glycans, whereas hFet predominantly biantennary glycan structures. The dominant O-glycans in all fetuins are of the core 1 mucin-type harboring one or two sialic acids. (c) The final step, proteolytic processing and phosphorylation, likely happens after glycosylation. The signal peptides are removed from the preproteins. For hFet, some unknown proteinase cleaves the C-terminal arginine at position 322 and converts hFet into a two-chain polypeptide form as we describe in more detail in Figure S3. Interestingly, we identified both the single-chain and two-chain polypeptide forms in rhFet, suggesting that in the recombinant expression system the cleavage of arginine is incomplete (Figure S4). For the phosphorylation, we found that all proteoforms of the hFet are fully monophosphorylated, whereas the phosphosite occupancy on bFet was about 25% monophosphorylated, 12% doubly phosphorylated, and 63% nonphosphorylated. On rhFet we found no evidence at all for protein phosphorylation.
Figure 5
Figure 5
Fully annotated zero charge deconvoluted native mass spectrum of hFet. The overall PTM compositions were assigned based on the accurate mass measurements of the intact protein proteoforms. All proteoforms contain 1 phosphate moiety. The number of sialic acids attached is marked at the top of each peak. For example, the most abundant peak is marked in blue and number 8, as it corresponds to the glycan composition HexNAc11Hex13Neu5Ac8 and one phosphate moiety.
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