Netrin-1 can be a useful early diagnostic biomarker of acute kidney injury (AKI) after renal
transplantation. The use of netrin-1 in clinical practice requires that this biomarker be associated with
an analytical method that combines specificity, accuracy and robustness. This study aimed to develop
an optimized multiple reaction monitoring (MRM) method using ultrafast liquid chromatography coupled
with tandem mass spectrometry to measure urinary netrin-1 levels in renal transplant recipients.
Purified recombinant human netrin-1 tryptic standard was analyzed by Matrix-Assisted Laser Desorption
Ionization-Time of Flight Mass Spectrometry (MALDI-TOF) MS/MS and LC-MS/MS to select for peptides that provided
specificity and adequate response in developing an MRM method for urinary netrin-1 quantification. Human urine
samples collected from kidney transplant recipients were isolated, concentrated, precipitated and trypsin digested before
mass spectrometric analysis of netrin-1. Netrin-1 levels were also measured in urine samples by enzyme immunoassay.
The tryptic peptide ion MH+2 of 270DSYFYAVSDLQVGGR284 (m/z 839) provided an adequate signal and was used for
quantification of netrin-1 under conditions employed for LC-MS/MS analysis. MALDI-TOF MS/MS spectra obtained by
collision-induced dissociation of the parent MH+2 ion 270DSYFYAVSDLQVGGR284 resulted in y8, y9 and y11 product
ions that were used for quantitative analysis by MRM method. Urinary Netrin-1 content measured by LC-MS/MS after
transplantation was significantly higher compared to before transplantation levels. The Spearman correlation coefficient
between the two methods was statistically significant. Intra-day and inter-day coefficient of variation provided good repeatability
and reproducibility for validation of LC-MS/MS analysis. LC-MS/MS quantification of Netrin-1 may provide
a new reference method to determine changes of this potential biomarker in human kidney transplant patients.