Alpha-1-acid glycoprotein (AGP) is a highly glycosylated protein from human plasma with five N-type glycosylation sites carrying dominantly higher antennary structures and thus represents a challenging target for characterization of glycan heterogeneity. Here, we show that glycan composition over all five glycosylation sites can be determined quantitatively by ESI-qTOF-MS of the intact glycoprotein in negative ion mode. We find numerous glycan species extending the mass range of the glycoprotein species from 35.0 to 38.5 kDa. The dominant glycan compositions contain tri- and tetraantennary structures on all glycosylation sites. The mass degeneracy of two fucosyl units versus one sialic acid was resolved by treating the sample with sialidase and analyzing the resulting desialylated AGP by electrospray ionization-mass spectrometry in positive ion mode. The pattern of nonsialylated oligosaccharides was used for interpretation of the fully sialylated species using bioinformatics tools. From pooled human plasma, we find 90, 101, and 64 different glycan compositions for genetic variants ORM1*F1, ORM1*S, and ORM2, respectively. Glycan structures carry dominantly between 15 and 16 sialic acids indicating an almost complete termination of all antenae with sialic acid. AGP from human plasma samples of single individuals was analyzed as desialylated glycoproteins and showed variations in fucosylation and in the amount of antennary structures between individuals.

The structure of glycans from glycoproteins is highly relevant for their function. We tightly integrate liquid chromatography-mass spectrometry (LC-MS), MS/MS, and nuclear magnetic resonance (NMR) data to achieve a complete characterization of even isobaric glycans differing in only one linkage position or in the substitution in one branch. As example, we analyzed ten desialylated underivatized glycans from bovine fibrinogen. The molecules were separated on a PGC column, and LC-MS data allowed an assignment of the compositions of the glycans. MS/MS data of the same glycans allowed elucidation of sequence and to some extent of branching and linkage. All MS/MS fragmentation methods led to multiple dissociations, resulting in several cases in ambiguous data. The MS/MS data were interpreted both by scientists and automatically by software, and the differential results are compared. Additional data from a tight integration of LC-MS and NMR data resulted in a complete structural characterization of the glycans. The acquisition of simple 1D (1)H NMR data led--in combination with LC-MS and MS/MS data--to an unambiguous assignment of the isobaric glycans. Compounds that were not separated in the chromatography could easily be assigned structurally by applying the 3D cross-correlation (3DCC) technology to arrive at NMR spectra of the pure components-without actually separating them. By applying LC-MS, MS/MS, 1D (1)H NMR, and 3DCC together, one can assign glycan structures from glycoconjugates with high confidence affording only 200 pmol of glycan material.

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The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein syntaxin-5 (Stx5) is essential for Golgi transport. In humans, the STX5 mRNA encodes two protein isoforms, Stx5 Long (Stx5L) from the first starting methionine and Stx5 Short (Stx5S) from an alternative starting methionine at position 55. In this study, we identify a human disorder caused by a single missense substitution in the second starting methionine (p.M55V), resulting in complete loss of the short isoform. Patients suffer from an early fatal multisystem disease, including severe liver disease, skeletal abnormalities and abnormal glycosylation. Primary human dermal fibroblasts isolated from these patients show defective glycosylation, altered Golgi morphology as measured by electron microscopy, mislocalization of glycosyltransferases, and compromised ER-Golgi trafficking. Measurements of cognate binding SNAREs, based on biotin-synchronizable forms of Stx5 (the RUSH system) and Förster resonance energy transfer (FRET), revealed that the short isoform of Stx5 is essential for intra-Golgi transport. Alternative starting codons of Stx5 are thus linked to human disease, demonstrating that the site of translation initiation is an important new layer of regulating protein trafficking.

Protein N-glycosylation is a multifactorial process involved in many biological processes. A broad range of congenital disorders of glycosylation (CDGs) have been described that feature defects in protein N-glycan biosynthesis. Here, we present insights into the disrupted N-glycosylation of various CDG patients exhibiting defects in the transport of nucleotide sugars, Golgi glycosylation or Golgi trafficking. We studied enzymatically released N-glycans of total plasma proteins and affinity purified immunoglobulin G (IgG) from patients and healthy controls using mass spectrometry (MS). The applied method allowed the differentiation of sialic acid linkage isomers via their derivatization. Furthermore, protein-specific glycan profiles were quantified for transferrin and IgG Fc using electrospray ionization MS of intact proteins and glycopeptides, respectively. Next to the previously described glycomic effects, we report unprecedented sialic linkage-specific effects. Defects in proteins involved in Golgi trafficking (COG5-CDG) and CMP-sialic acid transport (SLC35A1-CDG) resulted in lower levels of sialylated structures on plasma proteins as compared to healthy controls. Findings for these specific CDGs include a more pronounced effect for α2,3-sialylation than for α2,6-sialylation. The diverse abnormalities in glycomic features described in this study reflect the broad range of biological mechanisms that influence protein glycosylation.