The sensitivity of oligosaccharides in mass spectrometry lags far behind that of peptides. This is a critical factor in realizing the high-throughput analysis of posttranslational modifications in proteomics. We here described that hydrazide derivatives of cyanine dyes (Cy3, Cy5) with a positive charge made excellent labeling reagents for the detection of oligosaccharides by matrix-assisted laser desorption/ionization mass spectrometry. Cy3-labeled standard N-glycan could be detected at 200 amol on the MALDI target plate in reflectron mode without any purification procedures after the labeling reaction, which may meet the level of sensitivity required in proteome research. Despite the general recognition that the production of signals of oligosaccharides under MALDI conditions would be highly dependent on the matrix, most of the known N-glycans from chicken ovalbumin could be detected upon Cye derivatization nearly independent of the kind of matrix tested (e.g., nor-harman, 2,5-dihydroxybenzoic acid and alpha-cyano-4-hydroxycinnamic acid) without spoiling the signal strength. Postsource decay afforded simple spectra mainly consisting of Y-type fragment ions, thus simplifying the sequence analysis. In-source decay afforded a similar fragmentation pattern only when acidic matrixes were used. In addition, this derivatization technique was successfully applied to the profiling of N-glycans of gel-separated glycoproteins.

Despite very similar tertiary structures based upon a common framework, legume lectins exhibit an amazing variety of sugar binding specificities. While most of these lectins recognize rather discrete sugar linkages, Phaseolus vulgaris erythroagglutinating and leukoagglutinating lectins (E 4 -and L 4 -PHA) are unique in recognizing larger structures. E 4 -and L 4 -PHA are known to recognize complex type N-glycans containing bisecting GlcNAc or a ␤1,6-linked branch, respectively. However, the detailed mechanisms of molecular recognition are poorly understood. In order to dissect the contributions of different portions of each lectin, we carried out region-swapping mutagenesis between E 4 -and L 4 -PHA. We prepared six chimeric lectins by exchanging different combinations of loop B and the central portion of loop C, two of four loops thought to be important for the recognition of monosaccharides (Sharma, V., and Surolia, A. (1997) J. Mol. Biol. 267, 433-445). The chimeric lectins' sugar binding activities were evaluated quantitatively by surface plasmon resonance. These comparisons indicate that the high specificities of E 4 -and L 4 -PHA toward bisecting GlcNAc and ␤1,6-linked branch structures are almost solely attributable to loop B. The contribution of the central portion of loop C to the recognition of those structural motifs was found to be negligible. Instead, it modulates affinity toward LacNAc residues present at the nonreducing terminus. Moreover, some of the chimeric lectins prepared in this study showed even higher specificities/affinities than native E 4 -and L 4 -PHA toward complex sugar chains containing either a bisecting GlcNAc residue or a ␤1,6-linked branch.The legume lectins are a family of sugar-binding proteins found mainly in the seeds of plants belonging to the Leguminosae family (1-3). Lectins from leguminous plants constitute a large family of homologous proteins displaying remarkable divergence in their carbohydrate specificity. Elucidation of the mechanism by which these lectins can possess such a broad range of binding specificities while maintaining a strikingly similar threedimensional monomer structure will be key to understanding the essence of carbohydrate-protein interactions.At present, the crystal structures of ϳ20 legume lectins have been solved.1 These various lectin monomers share a common so-called "jellyroll" structure composed primary of a six-and a seven-stranded antiparallel ␤-sheet. Each monomer binds a manganese and a calcium ion that are both essential for carbohydrate binding. Amino acid sequence comparisons, x-ray crystallographic analysis, and mutagenesis studies revealed that the differences in carbohydrate specificity appear to be due primarily to differences in amino acid residues residing in loops adjacent to the carbohydrate binding site.The primary carbohydrate-binding site of legume lectins is a shallow depression on loops associated with the concave face of the seven-stranded curved ␤-sheet (5). It is constructed mainly by residues from four sequentially se...