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A new method for the mass spectrometric characterization of site-specific protein glycosylation is presented. Glycoprotein samples were subjected to unspecific proteolysis by Pronase, resulting in glycopeptides with peptide moieties of mostly two to eight amino acids. Resulting (glyco-)peptide samples were resolved by nanoscale normal-phase liquid chromatography (LC)-online mass spectrometry (MS). Retention depended on the size of the glycan chain and allowed the separation of identical peptide moieties containing different N-glycan structures. Glycopeptides were analyzed in an ion trap instrument performing repetitive ion isolation/fragmentation cycles. While the MS/MS spectra were dominated by fragmentations of glycosidic linkages, MS(3) spectra exhibited cleavages of the peptide backbone and provided information on the peptide sequence and glycan attachment site. When applied to the model glycoproteins ribonuclease B and horseradish peroxidase (HRP), the method provided detailed insights into protein glycosylation and revealed some new features of site-specific glycosylation of HRP. Application of the method to Dolichos biflorus lectin, which has hitherto not been studied with respect to its glycosylation, identified two glycans attached alternatively to its single glycosylation site. Thus, the presented, unique combination of Pronase digestion of glycoproteins, normal-phase nano-LC, and multistage MS provides a method for the facile characterization of site-specific protein glycosylation.

Pulmonary surfactant protein (SP)-D is an important component of the innate immune system of the lung, which is thought to function by binding to specific carbohydrates on the surface of viruses and unicellular pathogens. SP-D has been shown to have a relatively high affinity for the monosaccharides mannose, glucose, and fucose. However, there is limited information on SP-D binding to complex carbohydrate structures, and binding of SP-D to fucose in the context of an oligosaccharide has not yet been investigated. In this study, we used surface plasmon resonance spectroscopy to examine the potential of SP-D to bind to various synthetic fucosylated oligosaccharides, and identified Fucalpha1-3GalNAc and Fucalpha1-3GlcNAc elements as strong ligands. These types of fucosylated glycoconjugates are presented at the surface of Schistosoma mansoni, a parasitic worm that, during development, transiently resides in the lung. In line with the findings by surface plasmon resonance, we found that SP-D can bind to larval stages of S. mansoni, demonstrating for the first time that SP-D interacts with multicellular lung pathogens.