Filiz Ozcan, Halide Akbas, Gultekin Suleymanlar, Mutay Aslan and Gultekin Yucel Pages 31 - 38 ( 8 )
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.
Netrin-1, renal transplantation, tandem mass spectrometry.
Akdeniz University Medical School, Department of Medical Biochemistry, 07070, Antalya, Turkey