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.

CHCA-modified Au nanoparticles for laser desorption ionization mass spectrometric analysis of peptides

“…The results show AuNPs can provide the capacity for ionization of more than one analyte per laser pulse. AuNPs have also been applied for SALDI-MS analysis of other samples, including aminothiols [17][18][19], triphosphate [19], small carbohydrates [20,21], as well as oligosaccharides [18]. The use of AuNPs in SALDI-MS offers several advantages, including ease of preparation and chemical modification, high absorption coefficient, independence from irradiation wavelength, and excellent stability and biocompatibility.…”

“…As certain molecules can be selectively captured on the nanoparticle surface, UV energy from the laser can be efficiently transferred for desorption and ionization of the analyte of interest. Therefore, molecules with poor ionization efficiency in traditional MALDI-MS, for instance saccharides, can be analyzed by cationization in SALDI-MS with AuNPs [18,20,21].…”

Structural and Functional Glycosphingolipidomics by Glycoblotting with an Aminooxy-Functionalized Gold Nanoparticle

“…Recent our results revealed that ionization of analytes is highly enhanced by specific laser-scattering or diffused reflection on the extremely increased surface area of metal 6 nanoparticles, which may greatly accelerate the ionization of chemisorbed small molecules from GNP 22,23 . We hypothesized that a streamlined protocol by integrating To illustrate the new method, crude lipids fraction 24 of C57BL/6JJcl adult (7 weeks) and embryonic (13.5 days) mouse brains were subjected directly to "ozonolysis" and "glycoblotting" protocol.…”

Glycosyltransferases and their Assays

“…Another attractive method for label-free enzyme assays is mass spectrometry, and several protocols have recently been published for mass spectrometry-based GT assays [82][83][84] (Figure 2 A). Davis and co-workers, for example, have employed mass spectrometry for the kinetic characterisation and HTS of two GTs, bovine b-1,4-GalT and the glucosyltransferase UGT72B1 from A. thaliana.…”