MSACL 2019 EU Abstract

MALDI Chip Technology for in situ Detection of Human Procalcitonin

Josef Dvorak (2), Michael Volny (1), Petr Novak (1,2) and Petr Pompach (1,2,3)
(1) Institute of Microbiology, v.v.i., Czech Academy of Sciences, Prague, Czech Republic (2) Faculty of Science, Charles University, Prague, Czech Republic (3) Institute of Biotechnology, v.v.i., Czech Academy of Sciences, Vestec, Czech Republic

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Petr Pompach (Presenter) Institute of Microbiology

Relevant Financial Disclosures (within past 24 months) Grant/Research Support Ministry of Health (project NV19-05-00541) Committee/Board/Advisory Board Czech Society for Mass Spectrometry Salary Institute of Biotechnology Discussing Products? NO, presenter will NOT present educational content related to products/services of the ineligible company(ies) listed above. Conflict mitigation NOT REQUIRED.

INTRODUCTION: Procalcitonin (PCT) is a 114 amino acids precursor of calcitonin produced by thyroidal C-cells and other neuroendocrine cells. PCT is clinically well recognized acute-phase protein biomarker, whose levels increase within 4 to 12 h upon stimulation. Compared to other sepsis markers, PCT could distinguish between infectious and non-infectious systematic inflammation or between viral and bacterial infections. Serum concentrations of PCT can vary from 0.1 ng/mL in healthy individuals to 120 ng/mL in patients with acute septic shock. In this study we show a technology for fast in-situ enrichment and mass spectrometry detection of PCT in human serum. The presented technology allows preparation of MALDI compatible protein chips by ambient ion landing. The electrosprayed proteins immobilized on conductive surfaces allow a wide range of bioanalytical assays. Compare to other materials, non-reactive surfaces suffer minimal nonspecific interactions with chemical species in the investigated sample and are thus an ideal substrate for selective protein chips. The reaction takes place directly on the protein chip and is followed by in-situ analysis by MALDI mass spectrometry.

OBJECTIVES: The objective of this study was to identify a low abundant PCT using in-situ MALDI chip technology.
METHODS: For detection of PCT, MALDI chips were prepared by functionalization of indium-tin-oxide glass using ambient ion landing of electrosprayed anti-procalcitonin antibody. Because of low levels of PCT in human serum, fast pre-concentration procedure based on acetonitrile precipitation was used before in-situ enrichment of PCT on MALDI chips. One microliter of fraction containing pre-concentrated PCT was applied on the antibody-modified spots. After incubation, washing and matrix deposition, the PCT molecule was detected by MALDI-TOF mass spectrometer.

RESULTS: The effectivity of pre-concentration step to enrich PCT was tested by western blot analysis. The optimized ratio of serum/water/acetonitrile was 1:2:5 (v/v). The pre-concentration step enriched the PCT protein by factor of 30. The modified MALDI chips further selectively enriched the PCT molecule, which was observed in the MALDI spectrum as singly and doubly charged ion at m/z 14000 (1+) and at m/z 7000 (2+). The achieved limit of detection for PCT was 10 ng/mL with S/N ratio 14 for the doubly charged ion.

CONCLUSION: The results show the possibility to detect clinically important antigens at very low concentration levels by using combination of simple pre-concentration step, in-situ enrichment on functionalized surfaces and MALDI mass spectrometry. This opens the door to immunoMALDI assays that allow sensitive determination of antigens in serum and could lead to fast clinical assays that can be easily automated for high-throughput screening.