MSACL 2019 EU Abstract

Monitoring of Human Procalcitonin by Functionalized MALDI Surfaces

Josef Dvorak (1), Petr Novak (1,2), Petr Pompach (1,2,3)
(1) Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic (2) Institute of Microbiology, The Czech Academy of Sciences, Vestec, Czech Republic (3) Institute of Biotechnology, The Czech Academy of Sciences, Vestec, Czech Republic

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Josef Dvorak (Presenter) Department of Biochemistry, Charles University

Relevant Financial Disclosures (within past 24 months) Salary Institute of Microbiology Conflict mitigation NOT REQUIRED.

INTRODUCTION:
Increase of procalcitonin (PCT) concentration in human bloodstream is an valuable biomarker of sepsis. There are several commercialy available assays for determinination of concentration level of PCT. Our approach combines in-situ enrichment of procalcitonin by functionalized surfaces and MALDI mass spectrometry. The novel method might be fast, robust and low-cost alternative to standard approaches with a benefit of individual PCT forms determination.
OBJECTIVES:
Our interest is the application of functionalized MALDI surfaces for clinical analysis of PCT in human serum. Its concentration in bloodstream is under physiological conditions bellow the limit of detection of current methods. During sepsis, its concentration increases dramatically and PCT can be monitored.
METHODS:
Human serum samples with PCT were pre-concentrated using acetonitrile in a ratio of serum/water/acetonitrile 1:2:4.5 (v/v). Aqueous phase was dried and re-suspended in LC-MS grade water (5 µl). Two microliters of the sample were incubated on MALDI chip functionalized with anti-procalcitonin antibody for 60 minutes in humidity chamber. After incubation, the MALDI chip was washed three times in PBS, rinsed in distilled water and let dried at RT. Dihydroxyacetophenone matrix was applied on each spot. Procalcitonin detection was performed by Autoflex speed MALDI – TOF MS (Bruker Daltonics) in linear positive mode.
RESULTS:
Low concentration of PCT in human serum does not allow its direct detection by functionalized MALDI surfaces. Several workflows for PCT pre-concentration were optimized and successfully applied to precipitate the majority of serum proteins while keeping PCT in aqueous phase. By using this pre-concentration step followed by in-situ enrichment of PCT on functionalized surfaces, we were able to detect PCT at a concentration level of 0.5 fmol/μl (10 ng/ml).
CONCLUSION:
Our data suggests that imunoaffinity surfaces in combination with MALDI mass spectrometry are potentionally useful for detection of PCT in human serum. Further optimization of the technology and workflow is needed.