Abstract
Congenital disorders of glycosylation (CDGs) are a family of N-linked glycosylation defects associated with severe clinical manifestations. In CDG type-I, deficiency of lipid-linked oligosaccharide assembly leads to the underoccupancy of N-glycosylation sites on glycoproteins. Although the level of residual glycosylation activity is known to correlate with the clinical phenotype linked to individual CDG mutations, it is not known whether the degree of N-glycosylation site occupancy by itself correlates with the severity of the disease. To quantify the extent of underglycosylation in healthy control and in CDG samples, we developed a quantitative method of N-glycosylation site occupancy based on multiple reaction monitoring LC-MS/MS. Using isotopically labeled standard peptides, we directly quantified the level of N-glycosylation site occupancy on selected serum proteins. In healthy control samples, we determined 98-100% occupancy for all N-glycosylation sites of transferrin and alpha(1)-antitrypsin. In CDG type-I samples, we observed a reduction in N-glycosylation site occupancy that correlated with the severity of the disease. In addition, we noticed a selective underglycosylation of N-glycosylation sites, indicating preferential glycosylation of acceptor sequons of a given glycoprotein. In transferrin, a preferred occupancy for the first N-glycosylation site was observed, and a decreasing preference for the first, third, and second N-glycosylation sites was observed in alpha(1)-antitrypsin. This multiple reaction monitoring LC-MS/MS method can be extended to multiple glycoproteins, thereby enabling a glycoproteomics survey of N-glycosylation site occupancies in biological samples.
Highlights
Congenital disorders of glycosylation (CDGs) are a family of N-linked glycosylation defects associated with severe clinical manifestations
CDG type-I (CDG-I), CDG type-II (CDG-II), hereditary fructose intolerance (HFI), and healthy individuals were analyzed by transferrin isoelectric focusing gel electrophoresis, and the typical deviations of the band pattering of CDG sera from the healthy controls could be observed (Fig. 1)
In CDG-II, this pattern can be explained by altered trimming and elongation of transferrin N-glycan structures, which leads to decreased transfer of terminal sialic acid
Summary
Isoelectric Focusing Gel Electrophoresis—Serum transferrin was saturated for 30 min with ferric citrate (0.4 mM) in the presence of sodium hydrogen carbonate (20 mM) and separated on a rehydrated Immobiline DryPlate gel (GE Healthcare) with a pH range of 4.0 –7.0 using the PhastSystem (Amersham Biosciences). The reduced and alkylated samples were redissolved in 50 l of 20 mM ammonium bicarbonate, 10% ACN containing Asp-N and trypsin (0.5 g each, Roche Applied Science) and digested for 16 h at 37 °C. The samples were redissolved in 20 mM ammonium bicarbonate in 95 atom % H218O (Sigma), and 1.5 units of peptide N-glycanase F (PNGaseF; Roche Applied Science) reconstituted with 95 atom % H218O were added. The first 23 min, MRM transitions and product ion scans corresponding to the second, C-terminal transferrin N-glycosylation site were recorded (TFP2NVT, TFP2DVT, and corresponding sample peptides). Data corresponding to the first N-terminal transferrin N-glycosylation site were acquired for 25 min (TFP1NK, TFP1NKS, TFP1DK, TFP1DKS, and corresponding sample peptides). Data corresponding to the ␣1-antitrypsin N-glycosylation sites were acquired until completion of the LC run (ATP1N, ATP1D, ATP2N, ATP2D, ATP3N, ATP3D, and corresponding sample peptides). The percentage of site occupancy was calculated as the part of previously glycosylated, i.e. PNGaseF-sensitive, peptides
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