Investigation of new biomarkers in renal transplant recipients with proteomic methods

Renal transplantation is the best choice for the treatment of end-stage kidney disease. Renal allograft biopsy is the reference standard for diagnosis of rejection, however due to its invasive nature, new clinical and biochemical diagnostic markers allowing early prediction of rejection has been a priority in renal transplantation. Urinary liver-fatty acid binding protein (L-FABP) has been evaluated as a promising early biomarker of renal ischemia in human kidney transplant patients. Netrin-1 can be a useful early diagnostic biomarker of acute kidney injury (AKI) after renal transplantation. The use of L-FABP and netrin-1 in clinical practice requires that these biomarkers 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 (LC-MS/MS) to measure urinary L-FABP and netrin-1 levels in renal transplant recipients. Purified recombinant human L-FABP and netrin-1 tryptic standards were 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 L-FABP and netrin-1 quantification. Human urine samples collected from kidney transplant recipients were isolated, concentrated, precipitated and trypsin digested before mass spectrometric analysis of L-FABP and netrin-1. Netrin-1 and L-FABP levels were also measured in urine and serum samples by enzyme immunoassay. The tryptic peptide ion MH+ of 50FTITAGSK57 (m/z 824) provided an adequate signal and was used for quantification of L-FABP under conditions employed for LC-MS/MS analysis. MALDI-TOF MS/MS spectra obtained by collision-induced dissociation of the parent MH+ ion 50FTITAGSK57 resulted in an y3 product ion that was used for quantitative analysis by MRM method. Likewise, 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. Serum L-FABP and netrin-1 levels were evaluated via ELISA and LC-MS/MS methods. But there were not any difference between protein levels in serum samples before and after transplantation. Urinary L-FABP levels analyzed with ELISA was significantly increased after transplantation. Urinary L-FABP and netrin-1contents 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 L-FABP and netrin-1 may provide a new reference method to determine changes of this potential biomarker in human kidney transplant patients.