Mass spectrometry detection of monomeric renalase in human urine

Abstract

Renalase is a recently discovered secretory protein, which is suggested to play a role (which still remains elusive) in regulation of blood pressure. Earlier it was purified from urine of healthy volunteers by means of ammonium sulfate fractionation and subsequent affinity chromatography (Xu et al. (2005) J. Clin. Invest., 115, 1275). The resultant purified preparation of renalase contained 2 proteins with molecular masses of 35 and 67-75 kDa. The authors believed that the latter represents a dimerization (aggregation) product of the 35 kDa protein. In this study we have detected relanase in urinary samples of 2 of 6 volunteers only after immunoaffinity enrichment of urinary samples subjected to ammonium sulfate precipitation. Electrophoresis of the purified preparation also demonstrated the presence of 2 proteins with molecular masses of 35 and 66 kDa, respectively. Mass spectrometry analysis of these proteins identified 35 and 66 kDa proteins as renalase and serum albumin, respectively. Thus, our results do not support suggestion on formation of renalase dimers and they indicate that urinary renalase excretion significantly varies in humans.

A new method for quantitative determination of renalase based on mass spectrometric determination of a proteotypic peptide labelled with stable isotopes

“…Previous mass spectrometric studies confidently identified both recombinant renalase used as a reference and also intracellular and extracellular renalases . However, taking into consideration existing evidence for proteolytic processing of this protein during its translocation to the extracellular space, and the absence of the N‐terminal peptide (1–21) in urinary renalase, we have chosen the peptide EGDCNFVAPQGISSIIK, covering positions 100–116 for subsequent preparation of the synthetic analogue containing stable heavy isotopes.…”

“…Urinary samples (50 mL) were collected from 12 healthy volunteers (10 males, 2 females, mean age 34 ± 13 years) who gave informed consent for the use of their biomaterial in this study (see Table for details). Quantitative determination of renalase was performed with urinary samples, which were concentrated as described earlier . Briefly, urinary samples (50 mL from each volunteer) were centrifuged for 15 min at 4000 rpm and 4°C (Beckman TJ6 centrifuge, bucket rotor) to remove cells and supernatant proteins were then precipitated with ammonium sulfate (75% saturation at 4°C).…”

“…Thus there is a clear need for antibody‐free methods of renalase detection. Recently we successfully used mass spectrometry for qualitative detection of renalase in human urine and cultivated HEK293T cells . In the case of renalase detection in urine, ammonium sulfate precipitation provided a convenient approach for concentrating full‐length renalase up to detectable limits and eliminating possible renalase‐derived peptides …”

Construction of the Coding Sequence of the Transcription Variant 2 of the Human Renalase Gene and Its Expression in the Prokaryotic System

“…In accordance with results by Pandini et al [6] the target protein product (recombinant nRenalase1) was accumulated in Rosetta (DE3) as an insoluble form in inclusion bodies. The purified protein was not subjected to refolding after purification and was directly used for custom-made generation of polyclonal antibodies, which were successfully used for recent mass spectrometry detection of hRenalase1 in human urine [13]. Table 2 summarizes purification steps of both proteins.…”

“…Resultant full-length coding sequences of both hRenalase1 and hRenalase2 were used for expression of the recombinant proteins in E. coli Rosetta (DE3) cells, and hRenalase1 was then used for generation of polyclonal anti-renalase antibodies, which effectively interacted not only with the purified recombinant hRenalase1, but also with hRenalase1 in urine of volunteers [13]. Results of this study indicate that hRenalase2 also interacts with the polyclonal anti-renalase antibodies (Figure 6).…”