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Targeted LC-MS/MS for the evaluation of proteomics biomarkers in the blood of neonates with necrotizing enterocolitis and late-onset sepsis

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Abstract

Late-onset sepsis (LOS) and necrotizing enterocolitis (NEC) are severe life-threatening conditions for neonates. Accurate, early diagnosis and timely initiation of treatment are crucial. Non-specific overlapping clinical signs along with the non-sensitive/specific diagnostic tools set obstacles to speedy, trustful diagnosis including differential diagnosis. The objective of this study was to evaluate the potential of targeted LC-MS/MS proteomics in identifying diagnostic biomarkers of NEC or LOS. We conducted a prospective case-control study evaluating serum proteomics profiles of 25 NEC, 18 LOS, and an equal number of matched control neonates, over three sampling points. Eighty-three concatemers and synthetic peptides belonging to 47 protein markers of the two diseases were selected after thorough literature search. A novel selected reaction monitoring (SRM), LC-MS/MS method was developed for their analysis and evaluation as potential biomarkers. Multivariate and univariate statistical analyses highlighted significant proteins in differentiating LOS and NEC neonates and diseased from controls. Moreover, panels of proteins were tested for their ability to distinguish LOS from NEC and controls. We suggest two panels of three proteins each, exhibiting very high diagnostic value for LOS and excellent diagnostic performance at the critical LOS-NEC differentiation, reaching an AUC ROC value close to 1 (0.999). These panels constitute a valuable starting point for further validation with broader cohorts of neonates, aiming to improve the clinical practice.

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References

  1. Stoll BJ, et al. Trends in care practices, morbidity, and mortality of extremely preterm neonates, 1993-2012. JAMA. 2015;314(10):1039–51.

    Article  CAS  Google Scholar 

  2. Eaton S, Rees CM, Hall NJ. Current research in necrotizing enterocolitis. Early Hum Dev. 2016;97:33–9.

    Article  Google Scholar 

  3. Rossi P, Botgros R. Report on the expert meeting on neonatal and paediatric sepsis. EMA/477725/2010, 2010.

  4. Sharma R, Hudak ML. A clinical perspective of necrotizing enterocolitis: past, present, and future. Clin Perinatol. 2013;40(1):27–51.

    Article  Google Scholar 

  5. Stoll BJ, et al. Late-onset sepsis in very low birth weight neonates: the experience of the NICHD neonatal research network. Pediatrics. 2002;110(2 Pt 1):285–91.

    Article  Google Scholar 

  6. Shah J, Jefferies A, Yoon E, Lee S, Shah P, on behalf of the Canadian Neonatal Network. Risk factors and outcomes of late-onset bacterial sepsis in preterm neonates born at < 32 weeks’ gestation. Am J Perinatol. 2014;32(07):675–82.

    Article  Google Scholar 

  7. Bassler D, et al. Using a count of neonatal morbidities to predict poor outcome in extremely low birth weight infants: added role of neonatal infection. Pediatrics. 2009;123(1):313–8.

    Article  Google Scholar 

  8. Tepas JJ, et al. Trajectory of metabolic derangement in infants with necrotizing enterocolitis should drive timing and technique of surgical intervention. J. Am. Coll. Surg. 2010;210(5):847–52, 852–854.

    Article  Google Scholar 

  9. Han YY, et al. Early reversal of pediatric-neonatal septic shock by community physicians is associated with improved outcome. Pediatrics. 2003;112(4):793–9.

    Article  Google Scholar 

  10. Niemarkt HJ, et al. Necrotizing enterocolitis: a clinical review on diagnostic biomarkers and the role of the intestinal microbiota. Inflamm Bowel Dis. 2015;21(2):436–44.

    Article  Google Scholar 

  11. Delanghe JR, Speeckaert MM. Translational research and biomarkers in neonatal sepsis. Clin Chim Acta Int J Clin Chem. 2015;451(Pt A):46–64.

    Article  CAS  Google Scholar 

  12. Cao Z, Robinson RAS. The role of proteomics in understanding biological mechanisms of sepsis. Proteomics Clin Appl. 2014;8(1–2):35–52.

    Article  CAS  Google Scholar 

  13. Buhimschi CS, et al. Using proteomics in perinatal and neonatal sepsis. Hopes and challenges for the future. Curr Opin Infect Dis. 2009;22(3):235–43.

    Article  CAS  Google Scholar 

  14. Jiang P, Siggers JLA, Ngai HH-Y, Sit W-H, Sangild PT, Wan JM-F. The small intestine proteome is changed in preterm pigs developing necrotizing enterocolitis in response to formula feeding. J Nutr. 2008;138(10):1895–901.

    Article  CAS  Google Scholar 

  15. Cao Z, Yende S, Kellum JA, Angus DC, Robinson RAS. Proteomics reveals age-related differences in the host immune response to sepsis. J Proteome Res. 2014;13(2):422–32.

    Article  CAS  Google Scholar 

  16. Ng PC, et al. Host-response biomarkers for diagnosis of late-onset septicemia and necrotizing enterocolitis in preterm infants. J Clin Invest. 2010;120(8):2989–3000.

    Article  CAS  Google Scholar 

  17. Stewart CJ, et al. Metabolomic and proteomic analysis of serum from preterm infants with necrotising entercolitis and late-onset sepsis. Pediatr Res. 2016;79(3):425–31.

    Article  CAS  Google Scholar 

  18. Sylvester KG, et al. Urine protein biomarkers for the diagnosis and prognosis of necrotizing enterocolitis in infants. J. Pediatr. 2014;164(3):607–612.e1–7.

    Article  CAS  Google Scholar 

  19. Sylvester KG, et al. A novel urine peptide biomarker-based algorithm for the prognosis of necrotising enterocolitis in human infants. Gut. 2014;63(8):1284–92.

    Article  CAS  Google Scholar 

  20. Aebersold R, Burlingame AL, Bradshaw RA. Western blots versus selected reaction monitoring assays: time to turn the tables? Mol Cell Proteomics MCP. 2013;12(9):2381–2.

    Article  CAS  Google Scholar 

  21. Simpson DM, Beynon RJ. QconCATs: design and expression of concatenated protein standards for multiplexed protein quantification. Anal Bioanal Chem. 2012;404(4):977–89.

    Article  CAS  Google Scholar 

  22. Wolters JC, et al. Translational targeted proteomics profiling of mitochondrial energy metabolic pathways in mouse and human samples. J Proteome Res. 2016;15(9):3204–13.

    Article  CAS  Google Scholar 

  23. Buhimschi CS, et al. Proteomics mapping of cord blood identifies haptoglobin ‘switch-on’ pattern as biomarker of early-onset neonatal sepsis in preterm newborns. PloS One. 2011;6(10):e26111.

    Article  CAS  Google Scholar 

  24. Shen Z, et al. Sepsis plasma protein profiling with immunodepletion, three-dimensional liquid chromatography tandem mass spectrometry, and spectrum counting. J Proteome Res. 2006;5(11):3154–60.

    Article  CAS  Google Scholar 

  25. de Paiva RA, David CM, Domont GB. Proteomics in sepsis: a pilot study. Rev Bras Ter Intensiva. 2010;22(4):403–12.

    Article  Google Scholar 

  26. Soares AJC, Santos MF, Trugilho MRO, Neves-Ferreira AGC, Perales J, Domont GB. Differential proteomics of the plasma of individuals with sepsis caused by Acinetobacter baumannii. J Proteome. 2009;73(2):267–78.

    Article  CAS  Google Scholar 

  27. Kalenka A, Feldmann RE, Otero K, Maurer MH, Waschke KF, Fiedler F. Changes in the serum proteome of patients with sepsis and septic shock. Anesth Analg. 2006;103(6):1522–6.

    Article  CAS  Google Scholar 

  28. Gundry RL, et al. Preparation of proteins and peptides for mass spectrometry analysis in a bottom-up proteomics workflow. Curr Protoc Mol Biol. 2009; https://doi.org/10.1002/0471142727.mb1025s88.

  29. MacLean B, et al. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics. 2010;26(7):966–8.

    Article  CAS  Google Scholar 

  30. Xia J, Broadhurst DI, Wilson M, Wishart DS. Translational biomarker discovery in clinical metabolomics: an introductory tutorial. Metabolomics. 2013;9(2):280–99.

    Article  CAS  Google Scholar 

  31. Eriksson L, Byrne T, Johansson E, Trygg J, Vikström C. Multi- and Megavariate data analysis basic principles and applications: Umetrics Academy; 2013.

  32. Gilfillan M, Bhandari V. Biomarkers for the diagnosis of neonatal sepsis and necrotizing enterocolitis: clinical practice guidelines. Early Hum Dev. 2017;105:25–33.

    Article  CAS  Google Scholar 

  33. Yang Y-X, Li L. Identification of potential biomarkers of sepsis using bioinformatics analysis. Exp Ther Med. 2017;13(5):1689–96.

    Article  CAS  Google Scholar 

  34. Sammalkorpi K, Valtonen V, Kerttula Y, Nikkilä E, Taskinen MR. Changes in serum lipoprotein pattern induced by acute infections. Metabolism. 1988;37(9):859–65.

    Article  CAS  Google Scholar 

  35. Levels JHM, et al. Alterations in lipoprotein homeostasis during human experimental endotoxemia and clinical sepsis. Biochim Biophys Acta. 2007;1771(12):1429–38.

    Article  CAS  Google Scholar 

  36. van Leeuwen HJ, Heezius ECJM, Dallinga GM, van Strijp JAG, Verhoef J, van Kessel KPM. Lipoprotein metabolism in patients with severe sepsis. Crit Care Med. 2003;31(5):1359–66.

    Article  Google Scholar 

  37. Chien J-Y, Jerng J-S, Yu C-J, Yang P-C. Low serum level of high-density lipoprotein cholesterol is a poor prognostic factor for severe sepsis. Crit Care Med. 2005;33(8):1688–93.

    Article  CAS  Google Scholar 

  38. Kumaraswamy SB, Linder A, Åkesson P, Dahlbäck B. Decreased plasma concentrations of apolipoprotein M in sepsis and systemic inflammatory response syndromes. Crit Care Lond Engl. 2012;16(2):R60.

    Article  Google Scholar 

  39. Yildiz B, Ucar B, Aksit A, Aydogdu SD, Colak O, Colak E. Diagnostic values of lipid and lipoprotein levels in late onset neonatal sepsis. Scand J Infect Dis. 2009;41(4):263–7.

    Article  CAS  Google Scholar 

  40. Grion CMC, et al. Lipoproteins and CETP levels as risk factors for severe sepsis in hospitalized patients. Eur J Clin Investig. 2010;40(4):330–8.

    Article  CAS  Google Scholar 

  41. Sarafidis K, et al. Urine metabolomics in neonates with late-onset sepsis in a case-control study. Sci Rep. 2017;7:45506.

    Article  CAS  Google Scholar 

  42. Phua J, et al. Characteristics and outcomes of culture-negative versus culture-positive severe sepsis. Crit. Care. 2013;17(5):R202.

    Article  Google Scholar 

Download references

Acknowledgements

This research has been co-financed—via a program of State Scholarships Foundation (IKY)—by the European Union (European Social Fund (ESF)) and Greek national funds through the action entitled “Scholarships programme for postgraduate studies—2nd Study Cycle” in the framework of the Operational Programme “Human Resources Development Program, Education and Lifelong Learning” of the National Strategic Reference Framework (NSRF) 2014–2020.

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Authors and Affiliations

  1. School of Chemistry, Aristotle University of Thessaloniki, 54124, Thessaloniki, Greece

    Anastasia Chrysovalantou Chatziioannou & Georgios Theodoridis

  2. Department of Pharmacy, Analytical Biochemistry, University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands

    Justina Clarinda Wolters, Natalia Govorukhina & Rainer Bischoff

  3. Department of Pediatrics, Center for Liver, Digestive and Metabolic Diseases, University of Groningen, University Medical Center Groningen, 9713 AV, Groningen, The Netherlands

    Justina Clarinda Wolters & Jan Albert Kuivenhoven

  4. 1st Department of Neonatology, School of Medicine, Hippokration Hospital, Aristotle University of Thessaloniki, 54124, Thessaloniki, Greece

    Kosmas Sarafidis, Agathi Thomaidou & Charalampos Agakidis

Authors
  1. Anastasia Chrysovalantou Chatziioannou
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  2. Justina Clarinda Wolters
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  3. Kosmas Sarafidis
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  4. Agathi Thomaidou
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  5. Charalampos Agakidis
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  6. Natalia Govorukhina
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  7. Jan Albert Kuivenhoven
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  8. Rainer Bischoff
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  9. Georgios Theodoridis
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Contributions

Anastasia Chrysovalantou Chatziioannou, Kosmas Sarafidis, Georgios Theodoridis, Justina Clarinda Wolters, and Rainer Bischoff conceived and designed the study and contributed to the realization of the analysis. Anastasia Chrysovalantou Chatziioannou, Kosmas Sarafidis, Georgios Theodoridis, Rainer Bischoff, Justina Clarinda Wolters, and Jan Albert Kuivenhoven. contributed to the manuscript’s drafting. Anastasia Chrysovalantou Chatziioannou performed analytical proteomics experiments, statistical analysis, and prepared tables (except ESM Table S1) and figures. Kosmas Sarafidis, Agathi Thomaidou, and Charalampos Agakidis were responsible for the medical care of neonates, clinical data evaluation, and sampling. Justina Clarinda Wolters and Rainer Bischoff. supervised sample analysis and provided the infrastructure required. Justina Clarinda Wolters developed the applied targeted proteomics essays. Jan Albert Kuivenhoven and Natalia Govorukhina provided concatemers and synthetic peptides respectively for the targeted proteomics analysis. All authors reviewed the manuscript.

Corresponding authors

Correspondence to Rainer Bischoff or Georgios Theodoridis.

Ethics declarations

The study protocol was reviewed and approved by the School of Medicine ethics committee of the Aristotle University of Thessaloniki. Informed written consent was obtained from all parents/guardians before enrolling the neonates in the study.

Conflict of interest

All authors declare that they have no conflict of interest.

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Chatziioannou, A.C., Wolters, J.C., Sarafidis, K. et al. Targeted LC-MS/MS for the evaluation of proteomics biomarkers in the blood of neonates with necrotizing enterocolitis and late-onset sepsis. Anal Bioanal Chem 410, 7163–7175 (2018). https://doi.org/10.1007/s00216-018-1320-3

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  • DOI: https://doi.org/10.1007/s00216-018-1320-3

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