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

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

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

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

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

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

Parsimonious Charge Deconvolution for Native Mass Spectrometry

Charge deconvolution infers the mass from mass over charge (<i>m</i>/<i>z</i>) measurements in electrospray ionization mass spectra. When applied over a wide input <i>m</i>/<i>z</i> or broad target mass range, charge-deconvolution algorithms can produce artifacts, such as false masses at one-half or one-third of the correct mass. Indeed, a maximum entropy term in the objective function of MaxEnt, the most commonly used charge deconvolution algorithm, favors a deconvolved spectrum with many peaks over one with fewer peaks. Here we describe a new “parsimonious” charge deconvolution algorithm that produces fewer artifacts. The algorithm is especially well-suited to high-resolution native mass spectrometry of intact glycoproteins and protein complexes. Deconvolution of native mass spectra poses special challenges due to salt and small molecule adducts, multimers, wide mass ranges, and fewer and lower charge states. We demonstrate the performance of the new deconvolution algorithm on a range of samples. On the heavily glycosylated plasma properdin glycoprotein, the new algorithm could deconvolve monomer and dimer simultaneously and, when focused on the <i>m</i>/<i>z</i> range of the monomer, gave accurate and interpretable masses for glycoforms that had previously been analyzed manually using <i>m</i>/<i>z</i> peaks rather than deconvolved masses. On therapeutic antibodies, the new algorithm facilitated the analysis of extensions, truncations, and Fab glycosylation. The algorithm facilitates the use of native mass spectrometry for the qualitative and quantitative analysis of protein and protein assemblies