A new method for quantitative determination of renalase based on mass spectrometric determination of a proteotypic...

Renalase is a recently discovered kidney secretory protein, which is considered as an important component involved in blood pressure regulation. Although altered levels of renalase have been detected in plasma and urine of patients with various kidney diseases, there is certain inconsistency of changes in the renalase levels reported by different laboratories. The latter is obviously associated with the use of the ELISA as the only available approach for quantitative analysis of renalase. Thus there is a clear need for the development of antibody-independent approaches for renalase quantification.

We have developed a new method for quantitative determination of human renalase, which is based on mass spectrometric detection of a proteotypic peptide containing С-terminal ¹³ C¹⁵ N-labelled lysine. It corresponds to a tryptic peptide of human renalase, which has been previously detected in most mass spectrometric determinations of this protein.

Using the labelled peptide H-EGDCNFVAPQGISSIIK-OH, corresponding to positions 100-116 of the human renalase sequence, as an internal standard and recombinant human renalase we have generated a calibration curve, which covered the concentration range 0.005-50 ng/mL with a limit of quantitation of 5 pg/mL. Using this calibration curve we were able to detect urinary renalase only after enrichment of initial urinary samples by ammonium sulfate precipitation (but not in untreated urine).

Results of our study indicate that quantitative determination of renalase based on mass spectrometric detection of a proteotypic peptide labelled with stable isotopes gives significantly lower values of this protein in human urine than those reported in the literature and based on the ELISA.