Human prorenin determination by hybrid immunocapture liquid chromatography/mass spectrometry: A mixed-solvent-triggered digestion utilizing D-optimal design.

RATIONALE: Human prorenin, representing the precursor of mature renin, has been discussed as a potential biomarker, e.g. in diagnosing primary hyperaldosteronism or diabetes-induced nephropathy. Currently, only immunoassays are available for prorenin quantification. As the similarity of prorenin to active renin impedes its accurate determination by immunoassay, mass spectrometry appears as an accurate alternative for differentiation of that protein.

METHODS

Immunoaffinity purification plus a mixed-solvent-triggered digestion was combined with liquid chromatography/mass spectrometry (LC/MS) to enable a fast, sensitive, and less laboratory-intensive approach to the quantification of prorenin. Statistical experimental planning, which is known as Design of Experiments (DOE), was used to identify the optimal conditions for the generation of the signature peptides within a manageable number of experiments. The efficiency of the mixed-solvent-triggered digestion by trypsin was investigated using four different organic solvents: acetonitrile, acetone, tetrahydrofuran and methanol.

RESULTS

By utilizing a D-optimal design, we found that the optimal mixed-solvent type for the generation of both signature peptides was acetonitrile at a concentration of 84% and an incubation temperature of 16°C. Using the mixed-solvent-triggered digestion, the procedure time allowed a fast analysis of active renin and prorenin with a short digestion time of 98 min. This optimized mixed-solvent-triggered digestion procedure was applied to detect renin and prorenin successfully in human plasma by the newly developed hybrid approach.

CONCLUSIONS

The identification of unique surrogates for human prorenin enabled the mass spectrometric differentiation between the two similar proteins. The novel hybrid approach successfully proved its ability to purify, detect and distinguish between prorenin and active renin in human plasma.