title: Direct and efficient monitoring of glycosyltransferase reactions on gold colloidal nanoparticles by using mass spectrometry.
abstract: A simple and efficient assay for glycosyltransferase activity on gold colloidal nanoparticles (GCNPs) by using laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS) is demonstrated by the enzymatic synthesis of the Lewis X trisaccharide on GCNPs containing GlcNAc residues. GCNPs containing multivalent sugars were well dispersed in aqueous solution and proved to be excellent acceptor substrates for the glycosyltransferase reaction. Direct LDI-TOF MS analysis of these GCNPs provided the ion peaks of the sugar derivatives, chemisorbed through S--Au linkages onto the GCNPs, even in the presence of contaminants such as proteins and salts. Thus, it enabled the rapid and direct detection of the enzymatic reaction on the GCNPs by subjecting a small amount (0.15 muL) of the reaction mixture to MS analysis without purification. Subsequent MS/MS analyses (LDI-LIFT-TOF/TOF method) of the product-carrying GCNPs enabled the structures of the sugar derivatives that had been constructed on the GCNPs by enzymatic glycosylation to be determined. A quantitative inhibition assay for glycosyltransferase by using LDI-TOF MS analysis on the GCNPs was demonstrated by using uridine 5'-diphosphate (UDP) as the inhibitor. This simple assay was then applied to the detection of the enzymatic activity of a crude cell extract of Escherichia coli, which produces Neisseria meningitidis beta-1,4-galactosyltransferase (beta-1,4-GalT). In this case, the GCNPs were roughly purified by means of ultrafiltration to remove the buffer and detergents before MS analysis. That the GCNPs are dissolved in solution in the reaction medium but are solid in the purification process is greatly advantageous for the simple and efficient detection of enzymatic activity in crude biological samples. Thus, GCNPs containing a variety of biomolecules may become a versatile and efficient tool for the rapid and direct monitoring of metabolism (metabolomics) in living cells when combined with LDI-TOF MS analysis.
authors.affiliation: Graduate School of Advanced Life Science, Frontier Research Center for the Post-Genome Science and Technology, Hokkaido University, N21, W11, Sapporo 001-0021, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Nagahori
authors.forename: Shin-Ichiro
authors.initials: S
authors.lastname: Nishimura
mesh_terms: D000117:Acetylglucosamine / Q000737:chemistry / Q000378:metabolism; D000327:Adsorption; D002240:Carbohydrate Sequence; D003102:Colloids; D005647:Fucosyltransferases / Q000737:chemistry / Q000378:metabolism; D016695:Glycosyltransferases / Q000737:chemistry / Q000378:metabolism*; D006046:Gold / Q000737:chemistry*; D006801:Humans; D016256:Lewis X Antigen / Q000031:analogs & derivatives; D053768:Metal Nanoparticles / Q000737:chemistry*; D008969:Molecular Sequence Data; D009237:N-Acetyllactosamine Synthase / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014312:Trisaccharides / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry
chemical_list: D003102:Colloids; D016256:Lewis X Antigen; D011994:Recombinant Proteins; D014312:Trisaccharides; C110530:galactosyl-(1,4)-fucopyranosyl-(1,3)-N-acetylglucosamine; D006046:Gold; D016695:Glycosyltransferases; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase; D009237:N-Acetyllactosamine Synthase; D000117:Acetylglucosamine