Development of a xylosyltransferase-I-selective UPLC MS/MS activity assay using a specific acceptor peptide.

摘要: Abstract Xylosyltransferases-I and –II (XT-I –II) play an important role regarding the homeostasis of extracellular matrix. Both enzymes catalyze initial step proteoglycan (PG) biosynthesis by transfer xylose from their natural substrate uridine diphosphate (UDP) -xylose to a PG-core protein. The subsequent addition further sugars, catalyzed different glycosyltransferases, leads formation tetrasaccharide linker, which connects protein glycosaminoglycans. reason for appearance two XT isoforms in all higher organisms is not known remarkable, as both are able initiate PG biosynthesis. determination XT-I activity clinical importance because it can be used biomarker several PG-associated fibrotic diseases. Since previous assays did adequately differentiate between XT-isoforms, aim this study was develop selective mass spectrometric (MS) assay. For purpose, we initially isoform-specific supernatants successfully identify synthetic acceptor peptide xylosylated much more selectively when compared XT-II isoform. assay optimized concerning methodical parameters such injection volume incubation time reaction-mixture. By using samples covering broad XT-activity spectrum, validated only quantification cell culture but also human serum specimens. Compared previously assays, our newly developed test sensitive, less expensive easier perform high throughput.