Targeted LC-MS/MS platform for the comprehensive determination of peptides in the kallikrein-kinin system

Abstract

The kallikrein-kinin system (KKS) is involved in many physiological and pathophysiological processes and is assumed to be connected to the development of clinical symptoms of angioedema or COVID-19, among other diseases. However, despite its diverse role in the regulation of physiological and pathophysiological functions, knowledge about the KKS in vivo remains limited. The short half-lives of kinins, their low abundance and structural similarities and the artificial generation of the kinin bradykinin greatly hinder reliable and accurate determination of kinin levels in plasma. To address these issues, a sensitive LC-MS/MS platform for the comprehensive and simultaneous determination of the four active kinins bradykinin, kallidin, des-Arg(9)-bradykinin and des-Arg(10)-kallidin and their major metabolites bradykinin 2-9, bradykinin 1-7 and bradykinin 1-5 was developed. This platform was validated according to the bioanalytical guideline of the US Food and Drug Administration regarding linearity, accuracy, precision, sensitivity, carry-over, recovery, parallelism, matrix effects and stability in plasma of healthy volunteers. The validated platform encompassed a broad calibration curve range from 2.0–15.3 pg/mL (depending on the kinin) up to 1000 pg/mL, covering the expected concentrations in disease states. No source-dependent matrix effects were identified, and suitable stability of the analytes in plasma was observed. The applicability of the developed platform was proven by the determination of endogenous levels in healthy volunteers, whose plasma kinin levels were successfully detected in the low pg/mL range. The established platform facilitates the investigation of kinin-mediated diseases (e.g. angioedema, COVID-19) and enables the assessment of the impact of altered enzyme activities on the formation or degradation of kinins. Graphical abstract

Picomolar Sensitivity Analysis of Multiple Bradykinin-Related Peptides in the Blood Plasma of Patients With Hereditary Angioedema in Remission: A Pilot Study

“…Technological developments based on LC-MS/MS now supports the detection of several kinins with picomolar sensitivity and high reproducibility in biological fluids (12)(13)(14)(15)(16). The inclusion of biologically inactive fragments allows addressing the relative effects of various peptidases known to hydrolyze BK-related peptides.…”

“…Using a validated liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) platform of picomolar sensitivity developed for the analysis of eleven bradykininrelated peptides (12)(13)(14)(15)(16), we addressed the presence of BK-related peptides in the plasma of patients with HAE-C1INH and HAE-FXII during remission. We hypothesized that some of these peptides, including BK 1−5 , might be biomarkers of these forms of the disease, even if the buffering activity of kininases kept the patients symptom-free (kinin levels below a pharmacologic threshold for vascular effects).…”

Mass spectrometric study of variation in kinin peptide profiles in nasal fluids and plasma of adult healthy individuals

“…Both platforms are characterized by lower limits of quantification (LLOQ) down to 1.9 pM (depending on the kinin). Details on the assay characteristics have been published elsewhere [ 13 , 14 ]. All stated peptide concentrations were corrected for salt content and peptide purity, referring to the conducted amino acid analysis.…”

Reliable measurement of plasma kinin peptides: Importance of preanalytical variables

“…Recently, optimization of mass spectrometric sensitivity of kinins enabled the detection of several kinin peptides simultaneously. 20,21 However, despite a capable protease inhibitor stabilizing kinins, 22 kinin level variability persisted in preliminary experiments, signifying the influence of additional preanalytical variables. Subsequently, this study sought to develop a standardized procedure combining the findings from preanalytical blood specimen collection and handling and appropriate inhibition of kinin formation and degradation.…”