= Emerging. More than 5 years before clinical availability. (26.62%)
= Expected to be clinically available in 1 to 4 years. (38.91%)
= Clinically available now. (34.47%)
MSACL 2020 US : Burdman

MSACL 2020 US Abstract

Topic: Proteomics

Poster Presentation
Poster #64a
Attended on Wednesday at 10:00

The determination of human active renin and prorenin by LC-MS: the impact of injection solvent composition and material on precision and signal intensity

Ilja Burdman (Presenter)
Institut of Clinical Pharmacy and Pharmacotherapy


Authors: Ilja Burdman, Bjoern B. Burckhardt
Heinrich Heine University, Institute of Clinical Pharmacy and Pharmacotherapy, Dusseldorf, Germany


INTRODUCTION: Representing the extended renin-angiotensin-aldosterone system, active renin accompanied by its precursor prorenin plays a crucial role in physiological as well as pathological health states. In clinical analysis, active renin is used for diagnosis of hyperaldosteronism where the plasma levels have a low picogram per milliliter concentrations and might be affected by inaccurate measurement due to cross-reactivity of immunoassays with catalytical active prorenin. LC-MS is a reliable and selective alternative. Currently, no reliable LC-MS method is available for clinical analysis of active renin and prorenin. Subsequently, a hybrid assay was developed incorporating proteolytical digestion. However, in very low concentrations, the analysis suffers from poor repeatability affecting the precision of the assay. Since unspecific peptide adsorption has been identified as a major concern for this imprecision, a systematic investigation of the impact of the injection solvent composition and applied material on the reliable quantification of active renin and prorenin was performed. METHODS: A design of experiments approach was conducted for an effective and reliable estimation of the optimal injection solvent composition conjointly with the materials. Therefore, the tryptic digest of human prorenin and active renin was used to analyze various injection solvent compositions on five 96-well plates from four vendors (Waters, Eppendorf, Greiner and Brand). Different injection solvent compositions consist of variable concentrations of water (10%-98%), methanol (0%-40%), dimethylsulfoxide (1%-40%) and formic acid (1%-10%) were evaluated for each plate separately. The D-optimal model facilitated the identification of the best performing injection solvent composition characterized by the highest signal intensity and a high precision. The latter was performed by repeated sample analysis (in total 426 LC-MS runs). After analyzing each plate individually, a comparison of the individual optimal solvents was executed. The measurement of the dissolved digest was performed by using the pro-part signature peptide as a unique surrogate for prorenin and the mature-part signature peptide for active renin. By utilizing a Shimadzu Nexera UHPLC and a Phenomenex Kinetex XB-C18 column (1.7 µm 2.1x100 mm), the chromatographic separation was achieved and a Sciex TripleTOF 6600 HRMS was used for detection. RESULTS: This investigation of the different 96-well plates demonstrated the crucial impact of different materials and injection solvent compositions on signal intensity and precise determination of both peptides. Substantial different mixture of injection solvent composition were identified as optimal setting depending on the applied material (e.g. water content in the mixture ranged from 52-82% for mature-part signature peptide). Whereas the larger mature-part signature peptide had more robust properties regarding the repeatability in different injection solvents and plates (Waters QuanRecovery: 0.92; Waters regular: 0.95; Eppendorf protein low-bind: 0.89; Greiner: 0.97; Brand: 0.97), the smaller pro-part signature peptide was effected more crucially (Waters QuanRecovery: 0.78; Waters regular: 0.72; Eppendorf protein low-bind: 0.78; Greiner: 0.96; Brand: 0.95). The best performing plates were from Greiner and Brand which showed a high repeatability in all injection solvent mixtures and the highest precision. The most appropriate composition increased the mass spectrometric signal intensity by factor 3 compared to the initial mixture. CONCLUSION: The most suitable material and optimal injection solvent composition made endogenous human active renin and prorenin detectable on the LC-MS system by a substantial gain in sensitivity. Besides the higher intensity, a robust precision accomplished the pre-validation for a hybrid immunocapture LC-MS assay. After a full validation, active renin and prorenin will be assessable for clinical application.

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