Bradykinin (BK) is a 9-amino acid peptide hormone which is generated through proteolysis of Kinninogen. Further peptidase cleavage removes one (BK 1-8) or two (BK 1-7) C-terminal amino acids, and variable oxidation of the third Proline residue generates three more bradykinin variants. In tissue, alternative processing of kinninogen generates variants with an added N-terminal lysine (Lys-BK variants). BK and oxidized BK stimulate Bradykinin Receptor 2 (B2R), while BK 1-8 and oxidized BK 1-8 stimulate Bradykinin Receptor 1 (B1R), and BK 1-7 and further cleaved BK are believed to be inactive. B2R is broadly expressed and is involved in vasodilation, inflammation, and metabolic adaptation, while B1R is most important in the inflammation context. The ability to monitor BK variants in plasma, in real-time, using the developed assay allows investigation of this hormone's role in metabolism and it's utilization as a biomarker of inflammation in the context of post-surgery recovery.<br/><br/>We developed a rapid assay for measurement of oxidized and unmodified BK, BK (1-8), BK (1-7), and Lys-BK directly in diluted plasma. In the protocol, plasma is extracted from collected blood by centrifugation, spiked with stable isotope labeled BK, BK(1-8), and BK(1-7) standards, optionally frozen for storage, diluted 10-fold with 0.1% aqueous formic acid, and injected directly onto a Cyclone Turboflow trapping column (Thermo). The Turboflow column performs a low resolution size exclusion separation, removing proteins too large to enter the solid phase pores while also desalting the sample. Following loading, the bound peptides are separated by a reversed phase gradient and analyzed using scheduled parallel reaction monitoring (PRM) of the aforementioned BK variants. Method qualification showed that quantitation based on peak area ratio of endogenous BK to spiked standard is reproducible (CV &lt; 30% over 5 days) and the assay is linear in the 0.005 to 90 ug/uL concentration range. The analysis requires roughly 15 minute sample preparation and 15 minutes instrument analysis total (including a 5-minute cleaning and equilibration) and can thus be used in real-time to monitor plasma BK. The assay will be used to measure BK variants in various obesity cohorts to investigate its role in metabolism and in cardiac surgery patients at various time intervals to evaluate it's suitability as a marker of implant rejection.

Rapid quantitation of Bradykinin variants in plasma by LC-MS

ACS Fall 2021

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