BACKGROUND: Sepsis is a common worldwide health condition with high mortality. It is caused by a dysregulated immune response to the pathogen. Severe infections resulting in sepsis can be also determined by monitoring several bloodstream biomarkers, one of them being pro-hormone procalcitonin (PCT). PCT concentration in the bloodstream correlates well with sepsis and in severe cases increases up to a thousand times from the healthy physiological values in a short time. In this study, we developed a rapid technique for PCT detection by MALDI-TOF mass spectrometry, that uses in-situ enrichment directly on the specialized immuno MALDI chips that are utilized as MALDI plates. The method's ability to detect PCT was confirmed by comparing the results with LC-MS bottom-up workflow. The new method detects intact PCT by its m/z and uncovers its alternations in septic serum. METHODS: The MALDI chips used for the detection of PCT were prepared by ambient ion soft landing of anti-PCT antibody on an ITO glass slide. The chips were used for the development of the rapid MALDI-TOF MS method. A parallel method based on affinity enrichment on magnetic beads followed by LC-MS/MS data-dependent peptide microsequencing was used to prove PCT presence in the sample. All samples were also tested by ELISA to determine PCT concentration prior to analyzing them by mass spectrometry methods. RESULTS: The MALDI chip method was optimized using recombinant PCT spiked into the human serum. The PCT detection limit was 10 ng/mL. The optimized method was used to analyze 13 sera from patients suffering sepsis. The PCT results were confirmed by LC-MS/MS. The measurement of the intact PCT by the MALDI chip method revealed that sera of patients with severe sepsis have other forms of PCT present, which show post-processing of the primary sequence by cleavage of PCT, resulting in the formation of N and C termini fragments. CONCLUSIONS: Procalcitonin from human serum was successfully enriched and detected using immunoaffinity MALDI chips. The intact PCT was characterized in 13 septic patients. The method is more specific compared to non-MS-based immunoaffinity techniques and allows observation of different variants of PCT in septic patients.

Biomedical Center Faculty of Medicine in Pilsen Charles University Pilsen Czech Republic

Department of Biochemistry Faculty of Science Charles University Prague Czech Republic

Department of Internal Medicine Faculty of Medicine in Pilsen Pilsen University Hospital Charles University Pilsen Czech Republic

Institute of Biotechnology The Czech Academy of Science Prague Czech Republic

Institute of Microbiology The Czech Academy of Science Prague Czech Republic

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Cecconi M, Evans L, Levy M, Rhodes A. Sepsis and septic shock. Lancet. 2018;392(10141):75–87. doi: 10.1016/S0140-6736(18)30696-2. PubMed DOI

Fleischmann C, Scherag A, Adhikari NKJ, Hartog CS, Tsaganos T, Schlattmann P, et al. Assessment of global incidence and mortality of hospital-treated sepsis current estimates and limitations. Am J Respir Crit Care Med. 2016;193(3):259–72. doi: 10.1164/rccm.201504-0781OC. PubMed DOI

Singer M, Deutschman CS, Seymour C, Shankar-Hari M, Annane D, Bauer M, et al. The third international consensus definitions for sepsis and septic shock (sepsis-3) JAMA J Am Med Assoc. 2016;315:801–10. doi: 10.1001/jama.2016.0287. PubMed DOI PMC

Rhodes A, Evans LE, Alhazzani W, Levy MM, Antonelli M, Ferrer R, et al. Surviving sepsis campaign: international guidelines for management of sepsis and septic shock: 2016. Intensive Care Med. 2017;45(3):304–377. doi: 10.1007/s00134-017-4683-6. PubMed DOI

Kumar S, Tripathy S, Jyoti A, Singh SG. Recent advances in biosensors for diagnosis and detection of sepsis: a comprehensive review. Biosens Bioelectron. 2019;124–125:205–15. doi: 10.1016/j.bios.2018.10.034. PubMed DOI

Christ-Crain M, Müller B. Procalcitonin on the dusty way to the holy grail: a progress report. In: Vincent J-L, editor. Yearbook of intensive care and emergency medicine 2005. Berlin, Heidelberg: Springer-Verlag; 2005. pp. 461–76.

Becker KL, Nylén ES, White JC, Müller B, Snider RH. Procalcitonin and the calcitonin gene family of peptides in inflammation, infection, and sepsis: a journey from calcitonin back to its precursors. J Clin Endocrinol Metabol. 2004;89:1512–25. doi: 10.1210/jc.2002-021444. PubMed DOI

Weglöhner W, Struck J, Fischer-Schulz C, Morgenthaler NG, Otto A, Bohuon C, et al. Isolation and characterization of serum procalcitonin from patients with sepsis. Peptides. 2001;22(12):2099–103. doi: 10.1016/S0196-9781(01)00541-1. PubMed DOI

Vijayan AL, Ravindran S, Saikant R, Lakshmi S, Kartik R, Manoj G. Procalcitonin: a promising diagnostic marker for sepsis and antibiotic therapy. J Intensive Care. 2017;5(1):1–7. doi: 10.1186/s40560-017-0246-8. PubMed DOI PMC

Gregoriano C, Heilmann E, Molitor A, Schuetz P. Role of procalcitonin use in the management of sepsis. J Thorac Dis. 2020;12(Suppl 1):S5–15. doi: 10.21037/jtd.2019.11.63. PubMed DOI PMC

Jensen JU, Heslet L, Jensen TH, Espersen K, Steffensen P, Tvede M. Procalcitonin increase in early identification of critically ill patients at high risk of mortality. Crit Care Med. 2006;34(10):2596–602. doi: 10.1097/01.CCM.0000239116.01855.61. PubMed DOI

Samsudin I, Vasikaran SD. Clinical utility and measurement of procalcitonin. Clin Biochem Rev. 2017;38:59–68. PubMed PMC

Badu-Tawiah AK, Wu C, Cooks RG. Ambient ion soft landing. Anal Chem. 2011;83(7):2648–2654. doi: 10.1021/ac102940q. PubMed DOI

Tata A, Salvitti C, Pepi F. From vacuum to atmospheric pressure: a review of ambient ion soft landing. Int J Mass Spectrom. 2020;450:116309. doi: 10.1016/j.ijms.2020.116309. DOI

Krásný L, Pompach P, Strohalm M, Obsilova V, Strnadová M, Novák P, et al. In-situ enrichment of phosphopeptides on MALDI plates modified by ambient ion landing. J Mass Spectrom. 2012;47(10):1294–302. doi: 10.1002/jms.3081. PubMed DOI

Deng Z, Thontasen N, Malinowski N, Rinke G, Harnau L, Rauschenbach S, et al. A close look at proteins: Submolecular resolution of two- and three-dimensionally folded cytochrome c at surfaces. Nano Lett. 2012;12(5):2452–8. doi: 10.1021/nl3005385. PubMed DOI

Gologan B, Takáts Z, Alvarez J, Wiseman JM, Talaty N, Ouyang Z, et al. Ion soft-landing into liquids: Protein identification, separation, and purification with retention of biological activity. J Am Soc Mass Spectrom. 2004;15(12):1874–84. doi: 10.1021/jasms.8b02109. PubMed DOI

Pompach P, Benada O, Rosulek M, Darebná P, Hausner J, Ružička V, et al. Protein chips compatible with MALDI mass spectrometry prepared by ambient ion landing. Anal Chem. 2016;88(17):8526–8534. doi: 10.1021/acs.analchem.6b01366. PubMed DOI

Pompach P, Nováková J, Kavan D, Benada O, Růžička V, Volný M, et al. Planar functionalized surfaces for direct immunoaffinity desorption/ionization mass spectrometry. Clin Chem. 2016;62(1):270–8. doi: 10.1373/clinchem.2015.244004. PubMed DOI

Rosulek M, Darebna P, Pompach P, Slavata L, Novak P. Proteases immobilization for in situ time-limited proteolysis on MALDI chips. Catalysts. 2019;9(10):833. doi: 10.3390/catal9100833. DOI

Darebna P, Spicka J, Kucera R, Topolcan O, Navratilova E, Ruzicka V, et al. Detection and quantification of carbohydrate-deficient transferrin by MALDI-compatible protein chips prepared by ambient ion soft landing. Clin Chem. 2018;64(9):1319–1326. doi: 10.1373/clinchem.2017.285452. PubMed DOI

Krásný L, Pompach P, Strohalm M, Obsilova V, Strnadová M, Novák P, et al. In-situ enrichment of phosphopeptides on MALDI plates modified by ambient ion landing. J Mass Spectrom. 2012;47(10):1294–1302. doi: 10.1002/jms.3081. PubMed DOI

Perez-Riverol Y, Bai J, Bandla C, García-Seisdedos D, Hewapathirana S, Kamatchinathan S, et al. The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences. Nucleic Acids Res. 2022;50(D1):D543–52. doi: 10.1093/nar/gkab1038. PubMed DOI PMC

Ferreira CR, Yannell KE, Jarmusch AK, Pirro V, Ouyang Z, Graham CR. Ambient ionization mass spectrometry for point-of-care diagnostics and other clinical measurements. Clin Chem. 2016;62(1):99–110. doi: 10.1373/clinchem.2014.237164. PubMed DOI PMC

Pompach P, Benada O, Rosulek M, Darebná P, Hausner J, Ružička V, et al. Protein chips compatible with MALDI mass spectrometry prepared by ambient ion landing. Anal Chem. 2016;88(17):8526–34. doi: 10.1021/acs.analchem.6b01366. PubMed DOI

Rosulek M, Darebna P, Pompach P, Slavata L, Novak P. Proteases immobilization for in situ time-limited proteolysis on MALDI chips. Catalysts. 2019;9(10):833. doi: 10.3390/catal9100833. DOI

Darebna P, Spicka J, Kucera R, Topolcan O, Navratilova E, Ruzicka V, et al. Detection and quantification of carbohydrate-deficient transferrin by MALDI-compatible protein chips prepared by ambient ion soft landing. Clin Chem. 2018;64(9):1319–26. doi: 10.1373/clinchem.2017.285452. PubMed DOI

Hatzistilianou M. Diagnostic and prognostic role of procalcitonin in infections. Sci World J. 2010;10:1941–6. doi: 10.1100/tsw.2010.181. PubMed DOI PMC

Huynh HH, Bœuf A, Derbez-Morin M, Dupuy AM, Lalere B, Delatour V, et al. Development of an antibody-free ID-LC MS method for the quantification of procalcitonin in human serum at sub-microgram per liter level using a peptide-based calibration. Anal Bioanal Chem. 2021;413(19):4707–25. doi: 10.1007/s00216-021-03361-0. PubMed DOI

Proof-of-concept MALDI-TOF-MS assay for the detection of Toxin B enzymatic activity in Clostridioides difficile infection