Supplementary Material for: Biomarker Discovery by Mass Spectrometry in Cerebrospinal Fluid and Plasma after Global Hypoxia-Ischemia in Newborn Piglets

<b><i>Background:</i></b> Biomarkers may qualify diagnosis, treatment allocation, and prognostication in neonatal encephalopathy. Biomarker development is challenged by competing etiologies, inter-individual genetic variability, and a lack of specific neonatal markers. To address these challenges, we used a standardized neonatal hypoxic-ischemic (HI) encephalopathy model with pre- and post-HI sampling of cerebrospinal fluid (CSF) and plasma. <b><i>Objectives:</i></b> The study aimed to identify novel candidate protein biomarkers of HI encephalopathy in a newborn piglet model in CSF and plasma. <b><i>Methods:</i></b> F_iO₂ was lowered to 4% in 6 newborn piglets, then adjusted over a 45-min period keeping the amplitude integrated-EEG &lt; 7 µV to induce HI encephalopathy. CSF and plasma was sampled pre-HI and 2 h after HI, protein levels were then analyzed by mass spectrometry. <b><i>Results:</i></b> Protein levels after HI changed significantly for 18 CSF proteins and 37 plasma proteins. CSF and plasma data showed distinct information, although peptidyl-prolyl cis-trans isomerase A had elevated levels in both fluids. HI regulation involved functional groups such as the antioxidant system, cell proliferation, cell structure, and apoptosis. S100-A8, which increased the most in CSF (9.5 fold), is known to be involved in inflammatory and immune response and to be highly regulated during injury. In plasma, increased proteins included FABP1 (31.8 fold) and proteins with antioxidant (SOD1, GPX3) and lectin function (REG3A, LGALS3). <b><i>Conclusions:</i></b> In this exploratory study, we have identified candidate biomarkers for HI in CSF and plasma, many not previously associated with HI. Identified proteins are promising candidates for further validation in time series experiments and clinical studies.