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

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.