Versatile high resolution oligosaccharide microarrays for plant glycobiology and cell wall research.
Pedersen, H. L., Fangel, J. U., McCleary, B., Ruzanski, C., Rydahl, M. G., Ralet, M. C., Farkas, V., Von Schantz, L., Marcus, S. E., Andersen, M.C. F., Field, R., Ohlin, M., Knox, J. P., Clausen, M. H. & Willats, W. G. T. (2012). Journal of Biological Chemistry, 287(47), 39429-39438.
Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.
Tagging saccharides for signal enhancement in mass spectrometric analysis.
Chang, Y. L., Liao, S. K. S., Chen, Y. C., Hung, W. T., Yu, H. M., Yang, W. B., Fang, J. M., Chen, C. H. & Lee, Y. C. (2011). Journal of mass spectrometry, 46(3), 247-255.
MALDI-MS provides a rapid and sensitive analysis of large biomolecules such as proteins and nucleic acids. However, oligo- and polysaccharides are less sensitive in MS analysis partly due to their neutral and hydrophilic nature to cause low ionization efficiency. In this study, four types of oligosaccharides including aldoses, aminoaldoses, alduronic acids and α-keto acids were modified by appropriate tags at the reducing termini to improve their ionization efficiency. Bradykinin (BK), a vasoactive nonapeptide (RPPGFSPFR), containing two arginine and two phenylalanine residues turned out to be an excellent MS signal enhancer for maltoheptaose, GlcNAc oligomers and oligogalacturonic acids. In the MALDI-TOF-MS analysis using 2,5-dihydroxybenzoic acid (2,5-DHB) as the matrix, the GalA4–BK and GalA5–BK conjugates prepared by reductive amination showed the detection limit at 0.1 fmol, i.e. ∼800-fold enhancement over the unmodified pentagalacturonic acids. The remarkable MS enhancement was attributable to the synergistic effect of the basic arginine residues for high proton affinity and the hydrophobic property phenylalanine residues for facile ionization. A tetrapeptide GFGR(OMe) and an arginine linked phenylenediamine (H2N)2Ph-R(OMe) were thus designed to act as potent tags of oligosaccharides in MS analysis. Interestingly, concurrent condensation and lactonization of α2,8-linked tetrasialic acid (SA4) was carried out with (H2N)2Ph-R(OMe) to obtain a quinoxalinone derivative, which showed > 200-fold enhancement over unmodified SA4 in the MALDI-TOF-MS analysis.