Enzymatic Glycosylation of Peptide Arrays on Gold Surfaces

Post-translational modifications (PTM) of proteins occur in the secretory pathway of all eukaryotic organisms. For example, phosphorylation, acetylation, glycosylation, methylation and ubiquitinylation represent the major types of protein modifications.[1,2] Of these modifications, glycosylation is chemically the most complex, occurring either at an Asn residue (N-glycosylation)[3] in a conserved motif Asn–Xaa–Ser/Thr (in which Xaa can be any amino acid except Pro) or at a Ser/Thr residue (O-glycosylation) and playing a key role in the biosynthesis, folding, secretion and regulation of proteins.[4,5] Introduction of a β-d-glucosyl-N-acetyl group (β-GlcNAc) on a serine/threonine residue by O-N-acetyl-β-d-glucosaminyltransferases (OGT) in nuclear and cytoplasmic proteins compete with phosphorylation of the same site[6] and is associated with protein functional regulation or diseases such as cancer, Alzheimer's disease or diabetes.[7,8] The O-mannose-linked serine or threonine is common in yeast; fucose is essential in ABO blood-type antigen determination[9] and in Notch receptor signalling.[10] O-β-xylosyltransferases initiate proteoglycan biosynthesis.[11] Understanding the pattern and biological implications of post-translational glycosylation has been a major challenge, and in recent years the development of a platform for the general, fast and reliable identification of enzyme specificity has attracted considerable attention from researchers around the world.[12] Nagahori and Nishimura have developed a way of monitoring glycosyltransferase activity in solution on gold nanoparticles by using mass spectrometry.[13] Freire et al. have used surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF MS) for detecting enzymatic conjugation reactions in solution.[14] Whilst these studies have demonstrated the use of mass spectrometry as an excellent analytical tool, such enzyme assays in solution require additional purification/liquid handling steps and have a limited scope for further miniaturisation. The demands of high-throughput screening are better met by microarrays, in which the enzyme substrate remains immobilised on the array surface throughout enzymatic reaction and analysis. Here, we report such a (peptide) microarray platform for monitoring glycosyltransferases involved in protein O-glycosylation using the label-free MALDI-ToF MS analysis as a readout.[15,16]