Characterization of picomole amounts of oligosaccharides from glycoproteins by 1H NMR spectroscopy.

NMR spectroscopy is a very valuable tool in carbohydrate analysis. However, its use is limited if only small quantities of the sample are available. Literature data suggest that commercial probes are suitable for NMR spectroscopic analysis of samples in the range of a few nanomoles, which, for example, is equivalent to many micrograms of a decasaccharide. NMR spectroscopic characterization of oligosaccharides utilizing significantly less material is highly desirable for the analysis of glycan chains of biological origin, for example, glycoproteins. This would complement the analysis of oligosaccharides by mass spectrometry (MS) which is inherently more sensitive but provides less information. Here, we demonstrate that modern equipment can be used to record spectra of minute amounts of sugars, for example, sucrose or a complex N-type decasaccharide, down to 15 picomole. Special sample preparation techniques and instrument setup are required to record spectra at such low quantities. Importantly, water suppression by a factor of 500 000 has been achieved by utilizing a modified water suppression by “excitation sculpting”. This also makes it possible to observe signals only 50 Hz away from the solvent signal. Also, sample tube selection and preparation were optimized. Data were recorded with a 700 MHz NMR spectrometer equipped with a commercially available tripleresonance cryoprobe. Elucidation of the carbohydrate components of glycoproteins is a very important step in understanding the biological function of oligosaccharides. Although more than 60 % of the human proteome is thought to be glycosylated, the role of many glycan structures is not yet clear. Oligosaccharide chains attached to proteins can contribute to, for example, cell recognition, protein folding, and signal transduction. 4] Furthermore, it is known that dysglycosylation can cause severe diseases like the congenital disorders of glycosylation. Also, most cancer cells have an altered glycosylation pattern. This is currently the target for the development of vaccines and new diagnostic assays. Today mass spectrometry and NMR spectroscopy are the major techniques used to identify the structures of glycans. NMR spectroscopy is limited because its sensitivity is relatively low in comparison to that of mass spectrometry (MS). However, in the study of oligosaccharides, NMR spectroscopy is superior to MS as it offers information that cannot be obtained from MS, such as the determination of 1) the configuration of sugar residues that have the same molecular weight, 2) the anomeric configuration (a or b), 3) the target position of glycosidic linkages, 4) the position of substituents linked to the OH groups, like phosphates or sulfates, and 5) the position of functional groups other than OH. Currently it is believed that NMR characterization of molecules with commercial probes requires a few nanomoles of material. However, the development of cryoprobes improved sensitivity by a factor of 4, resulting in a reduction of acquisition time by a factor of 16. Here we show that a high-resolution 700 MHz NMR spectrometer equipped with a cryogenic probe can be used to record spectra of molecules down to a level of a few picomoles. This is of special importance as most compounds from biological sources are available in very limited quantities. Sucrose and a complex N-type decasaccharide were used as examples. The decasaccharide was characterized previously by Vliegenthart et al. using multidimensional NMR spectroscopy. It can be assigned easily from 1D NMR spectra using the structural reporter group concept (Figure 1). The lower limit of detection for a signal is defined as when its height is three times the root-mean-square of the noise. Following information from Bruker, in many cryoprobes the signal to noise ratio (S/N) measured in 3 mm tubes is about same as that in 5 mm tubes with the same concentration