Relevant Financial Disclosures
(within past 24 months)
No relevant financial relationship(s) to disclose.
Abstract
Introduction:
Procalcitonin(PCT) is employed as a specific biomarker for the diagnosis and monitoring of bacterial sepsis. To this day, the quantification of PCT is almost exclusively performed with immunological methods. Current LC-MS methods do not provide the necessary sensitivity to compete with these methods. Mass spectrometric methods can provide deeper insights into the PCT levels during sepsis.
Objectives:
The primary objective of the current project is the development and application of LC-MS/MS-based methods for the quantification of PCT in human blood using MRM-methodologies. An immune-based enrichment can provide the necessary increase in sensitivity to compete/verify with the established pure antibody-based methods. This method should perform comparable or better to current immuno-logical methods in terms of sensitivity and ruggedness.
Methods:
Recombinant PCT underwent a bead-based immuno-enrichment. Afterwards, PCT was released and cleaved into peptides by proteolytic cleavage. Trypsin, Chymo-trypsin, Asp-N, Glu-C and Thermolysin have been investigated for proteolysis. As an alternative workflow, on-bead digestion was considered. The absence of remaining PCT on the beads was controlled via western blot while the digestion kinetics were monitored with MALDI-TOF-MS (Autoflex Speed [Bruker, Billerica, USA]). The final quantification was carried out on an LC-MS/MS system (QTRAP 5500 LC-MS/MS system [AB Sciex, Redwood City, USA]).
Results:
Several proteases have been investigated, most promising results were obtained with trypsin. An LC-MS/MS method for PCT tryptic peptide and a western blot protocol for detection of PCT have been developed. Western blot will be used as an orthogonal method to support further LC-MS/MS method development. On-going work focusses on comparing different protocols for immune-affinity enrichment and PCT digestions. The immunoenrichment proved to lower the LLOQ in human serum by a factor of 15 to 5 ng/mL. Interestingly, trypsin digestion of PCT did show unexpected kinetic behavior in the processing of a tryptic PCT peptide. After an initial increase of the product concentration, no further digest could be observed after 5-10 min.
Conclusion:
PCT analysis at endogenous PCT levels is challenging and extensive method development is required. A combined Immunoenrichment-LC-MS/MS method has been developed with an LLOQ of 5 ng/mL. Further work is on-going to increase the sensitivity and the enrichment factor.
= Discovery stage.
= Translation stage.
= Clinically available.
