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2,5-Dihydroxybenzoic acid (DHB) is the matrix of choice for carbohydrate and glycopeptide analysis, but due to the inhomogeneous surface morphology of samples prepared with DHB, it is typically incompatible with automated measurements.
We describe a simple and rapid method for the analysis of glycoproteins, which combines (a) reducing the complexity of the digest mixtures with glyco-specific enrichment and (b) subsequent LC-MALDI-TOF MS/MS analysis with DHB as MALDI matrix. All samples were prepared on hydrophobic sample plates with hydrophilic anchors 400 or 600 μm in diameter confining the sample dimensions.
In a first step, the matrix was applied to the 384 sample spots (“anchors”) of a microtiter plate–shaped MALDI target. The LC eluate from CAP-RP-HPLC subsequently dissolved the DHB matrix confined to the hydrophobic boundaries of the anchors. Co-crystallization of glycopeptides in DHB suitable for the automated analysis was achieved.
This method was applied to recombinant human inte-grin alpha and beta; glycosylation sites were identified and described.
The MALDI-MSMS spectra of glycopeptides (N-linked type) include information about the structure of peptide moiety as well as the glycan part of the molecules. MALDI-TOF/TOF spectra permitted (a) the detection of N-linked glycopeptides by a neutral loss analysis across the entire LC-MALDI-MS/MS dataset, (b) the determination of the molecular weight of the pure peptide chain by typical fragmentation patterns, (c) the identification of the peptide part of the fragmented glycopeptide by means of simple database searching, and (d) initial information about the glycan composition and the attachment site.
LC-MALDI-TOF/TOF on DHB matrix preparation is a powerful approach for the detailed characterization of glycoproteins.