Measurement of Fibrosis Marker Xylosyltransferase I Activity by HPLC Electrospray Ionization Tandem Mass Spectrometry

Abstract

Background: Xylosyltransferase I (XT-I), the key enzyme in the biosynthesis of glycosaminoglycan chains in proteoglycans, has increased activity in the blood serum of patients with connective tissue diseases. Therefore, the measurement of serum XT-I activity is useful to monitor disease activity in these patients.

Human xylosyltransferases in health and disease

“…A novel, rapid, sensitive and specific HPLC (high-performance liquid chromatography) -electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method to assay xylosyltransferase activity in serum and other body fluids using the synthetic peptide biotin-NH-Gln-Glu-GluGlu-Gly-Ser-Gly-Gly-Gly-Gln-Lys-Lys(5-fluorescein)-CONH 2 as a xylosyltransferase acceptor was recently described (Fig. 3) [119]. The sample preparation of this liquid chromatography-tandem mass spectrometric (LC-MS/MS) xylosyltransferase activity assay includes simple incubation, heat denaturation and centrifugation steps, and was very easy and relatively fast in comparison with the radiochemical assays.…”

“…Validation of the LC-MS/MS xylosyltransferase activity assay was performed according to the requirements for bioanalytical method validations and diagnostic accuracy [120 -122]. The measurement of xylosylated peptide was linear over a wide range of concentration, and excellent sensitivity allowed for the precise quantification of serum xylosyltransferase, even in patients with severely reduced xylosyltransferase activities [119]. Due to the central role of xylosyltransferases in proteoglycan biology, the measurement of its enzyme activity in body fluids is of great interest.…”

“…Consequently, the search began for other marker proteins which are present in the peripheral blood and which can be quantified with high sensitivity and analytical precision. The properties of xylosyltransferases made them attractive targets as biochemical markers for the quantification of the actual proteoglycan biosynthesis rate: a) they catalyze the initial and rate-limiting step in the biosynthesis of glycosaminoglycans [25,100,101]; b) they are shed from the Golgi membrane and are secreted together with the proteoglycan in the extracellular space [19,25,42,87]; c) assays have been developed that allow the sensitive and precise determination of xylosyltransferase in body fluids [41] and that are also suitable for highthroughput analyses in routine diagnostics [119]. While the biological role of xylosyltransferase secretion is not understood, the quantification of xylosyltransferase activity in serum and other body fluids could be successfully validated as a marker of the proteoglycan biosynthesis rate (Table 1).…”

Human Xylosyltransferase II Is Involved in the Biosynthesis of the Uniform Tetrasaccharide Linkage Region in Chondroitin Sulfate and Heparan Sulfate Proteoglycans

“…Analysis of Xylosyltransferase Activity by HPLC Electrospray Ionization Tandem Mass Spectrometry-For the determination of xylosyltransferase activity by HPLC electrospray ionization tandem mass spectrometry, the xylosyltransferase-mediated incorporation of UDP-xylose into the synthetic peptide biotin-NH-QEEEGSGGGQKK(5-fluorescein)-CONH 2 was quantified as described recently (19). The characterization of the XT-I and XT-II reaction products was performed as described using the biotin-NH-QEEEGSGGGQKK(5-fluorescein)-CONH 2 as acceptor (9).…”

“…The bikunin-homologous peptide biotin-NH-QEEEGSGGGQKK(5-fluorescein)-CONH 2 (19) is the best substrate known so far for XT-IImediated xylosylation (K m 5.2 Ϯ 0.5 M). The Michaelis-Menten constant for recombinant bikunin was determined to be 22 Ϯ 2 M. The FGF-2-(1-24) peptide, which is homologous to the amino-terminal 24 amino acids of FGF-2 and which has been shown to be a good XT-I acceptor (29), is also recognized by XT-II (K m 62 Ϯ 21 M).…”