Development and validation of a liquid chromatography-triple quadrupole mass spectrometry method for the determination of isopeptide ε-(γ-glutamyl) lysine in human urine as biomarker for transglutaminase 2 cross-linked proteins

Lien Dejager*, Mark Jairaj, Kieran Jones, Timothy Johnson, Sherri Dudal, Yves Dudal, Patrick Shahgaldian, Rita Correro, Jun Qu, Bo An, Richard Lucey, Szabolcs Szarka, Robert Wheller, Alina Pruna, Sarah Kettell, Andrew Pitt, Paul Cutler

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

Determination of the levels of protein cross-linking catalysed by the activity of transglutaminase 2 in various disease states has remained a significant challenge. The ability to quantify the isopeptide ε-(γ-glutamyl) lysine, which can form as a heterogeneous bond within or between proteins has significant analytical and clinical potential as a biomarker in biofluids such as human urine. Increased transglutaminase 2 activity is associated with a number of diseases, such as fibrosis. Previously published methods have been based on classical amino acid analysis, however they require a complex multi-enzyme digestion in order to achieve complete protein digestion, whilst leaving the isopeptide cross link intact. These methods require high levels of enzymes, which contaminate the analysis and alter the dynamics of digestion. The amino acid analysis detection also lacked selectivity, especially where the levels of crosslink are expected to be low relative to the background protein levels. We have systematically addressed these challenges, by optimising the precipitation of the protein in urine, the use of innovative immobilised enzyme technology, which allows for efficient digestion without enzyme contamination and LC-MS/MS detection based on multiple reaction monitoring. This method was validated for its analytical performance characteristics, showing the method has a sensitivity of 0.1 ng/mL of ε-(γ-glutamyl) lysine in human urine with precision of less than 20% CV, and is selective as no interferences were observed that may adversely affect the analysis. As such this approach represents a significant advance in the ability to detect and quantify ε-(γ-glutamyl) lysine.

Original languageEnglish
Article number464002
Number of pages31
JournalJournal of Chromatography A
Volume1699
Early online date17 Apr 2023
DOIs
Publication statusPublished - 21 Jun 2023

Keywords

  • Transglutaminase 2
  • LC-MS/MS
  • urine
  • protein cross-linking
  • ε-(γ-glutamyl) lysine

Fingerprint

Dive into the research topics of 'Development and validation of a liquid chromatography-triple quadrupole mass spectrometry method for the determination of isopeptide ε-(γ-glutamyl) lysine in human urine as biomarker for transglutaminase 2 cross-linked proteins'. Together they form a unique fingerprint.

Cite this