Sample preparation comparison for the investigation of unstable peptidomic biomarkers of hypertension in human plasma

Introduction

The renin-angiotensin system (RAS) and kallikrein-kinin system (KKS) are a network of endogenous peptides with vasoconstrictive/vasodilative properties. They are associated with the development of hypertension, the most prevalent risk factor for cardiovascular diseases. However, they have not been studied extensively due to the instability of the peptides in human plasma.

Method development of the RAS and KKS in human plasma requires either protease inactivation or the use of a surrogate matrix. In the former, there is a background concentration of the endogenous peptides which may lead to inaccurate quantitation and in the latter, a surrogate matrix may not mirror the matrix effects in human plasma. The aim of this project was to develop and test different sample preparation methods for accurate quantification of the peptides in human plasma without the need for a surrogate matrix and develop a quantitative assay using liquid chromatography tandem mass spectrometry (LC-MS/MS).

Methods

Purchased human plasma were aliquoted into four 1.5 mL Eppendorf tubes and in the first tube, a protease inhibitor mix was added; the second aliquot was heated at 50 oC for 10 minutes and a protease inhibitor mix added after cooling; the third aliquot was only heated for 50 oC; the fourth aliquot was not heated and a protease inhibitor mix was not added.

Nine calibration standards ranging from 0.06 to 200 fmol/µL were prepared for the light and heavy RAS and KKS peptides in each of the four plasma aliquots to prepare to obtain a final sample volume of 100 µL. Four-hundred microlitres of acetonitrile was added to the samples, vortexed briefly and centrifuged for 10 mins at 20000 rpm. Two-hundred microlitres of the supernatant was dried down, lyophilised and reconstituted with 50µL of 5% acetonitrile, 0.1% formic acid. Six microlitres of the resulting solutions were injected into the Waters Xevo UPLC-MS/MS and the results from the chromatographic data analysed.

Results

The peptides had varying sensitivity and linearity in the four methods. With no heating/ addition of protease inhibitor mix, the peptides had low sensitivity and linearity, R2 < 0.67 and not all peptides were not detected (12/19 at LOD, 2.5 fmol/µL). Upon addition of protease inhibitor, the sensitivity and linearity of the assay increased, (19/19 at LOD = 0.125 fmol/µL), R2 > 0.93. With heating, the extraction method had high linearity however sensitivity did not improve, (19/19 at LOD = 0.125 fmol/µL), R2 > 0.95. Heating plus addition of protease inhibitor mix had the highest sensitivity and linearity for the peptides, (19/19 at LOD = 0.06 fmol/ µL), R2 > 0.98

In summary, we developed a novel assay for all endogenous angiotensin and kinin peptides in plasma. Assay linearity, and peptide stability was reserved upon heating and addition of protease inhibitor mix. Following validation, this assay has the potential to be utilised in the clinic for the management of patients with hypertension.