Separation technologies for glycomics.

Abstract

Progress in genome projects has provided us with fundamentals on genetic information; however, the functions of a large number of genes remain to be elucidated. To understand the in vivo functions of eukaryotic genes, it is essential to grasp the features of their post-translational modifications. Among them, protein glycosylation is a central issue to be discussed, considering the predominant roles of glycoproteins in cell-cell and cell-substratum recognition events in multicellular organisms. In this context, it is necessary to establish a core strategy for analyzing glycosylated proteins under the concept of the "glycome" [Trends Glycosci. Glycotechnol. 12 (2000) 1]. Though the term glycome should be defined, in analogy to the genome and proteome, as "a whole set of glycans produced in a single organism", here we propose a glycome project specifically focusing on glycoproteins. Principal objectives in the project are to identify: (1) which genes encode glycoproteins (i.e. genome information); (2) which sites among potential glycosylation sites are actually glycosylated (i.e. glycosylation site information); (3) what are the structures of glycans (i.e. structural information); and (4) what are the effects (functions) of glycosylation (functional information). For these purposes, two affinity technologies have been introduced. One is named the "glyco-catch method" to identify genes encoding glycoproteins [Proteomics 1 (2001) 295], and the other is the recently reinforced "frontal affinity chromatography" [J. Chromatogr. A 890 (2000) 261]. By the former method, genes that encode glycoproteins as well as glycosylation sites are systematically identified by the efficient combination of conventional lectin-affinity chromatography and contemporary in silico database searching. The following three actions have been devised for rapid and systematic characterization of glycans: (1) mass spectrometry to acquire exact mass information; (2) 2-D/3-D mapping to obtain refined chemical information; and (3) reinforced frontal affinity chromatography to determine affinity constants (K(a)-values) for a set of lectins. Pyridylaminated glycans are used throughout the characterization processes. In this review, the concept and strategy of glycomic approaches are described referring to the on-going glycome project focused on the nematode Caenorhabditis elegans.