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Comprehensive proteomic analyses require efficient and selective pre-fractionation to facilitate analysis of post-translationally modified peptides and proteins and automated analysis procedures for the detection, identification, and structural characterization of the corresponding peptide modification.
Selective capturing of glycopeptides and -proteins was attained by means of magnetic particles specifically functionalized with lectins or boronic acids that bind to various structural motifs. Human serum contains a high number of glycoproteins, comprising several orders of magnitude in concentration. Thereby, isolation and subsequent identification of low-abundant glycoproteins from serum is a challenging task.
Human serum was incubated with differentially functionalized magnetic micro-particles (lectins or boronic acids). Isolated proteins were released from the beads under acidic conditions, dried, and subsequently re-dissolved and digested with trypsin. The resulting complex mixture of peptides was subjected to LC-MALDI analysis. The respective glycoproteins were identified by direct MS/MS analysis and subsequent database searching. Intact glyco-peptides enriched by a second magnetic-bead purification on peptide level were directly subjected to LC-MALDI analysis to get structural information about the glycan and peptide parts. A precondition to this novel approach was the discovery of certain consensus peak patterns in the MALDI-MS/MS spectra, allowing the automatic determination of the peptide part and the glycosidic information of the glycopeptides supported by bioinformatics tools.
Applying this fast and simple approach, a high number of low-abundant proteins were identified, comprising known or predicted glycosylation sites. According to the specific binding preferences of the different types of beads, complementary results were obtained from experiments using magnetic ConA, LCA, WGA, jacalin, and boronic acid beads, respectively. The use of jacalin and boronic acid beads facilitates the enrichment of O-glycosidically modified proteins. In contrast, ConA, WGA, and LCA specifically bind N-glycosylated peptides and proteins. Few non-glycosylated proteins were identified, probably due to co-precipitation with glycosylated proteins.