Validation of Biomarkers for Risk Stratification in Sudden Cardiac Death in Cardiomyopathy

Sudden cardiac death (SCD) is globally recognized as a leading cause of unexpected out of hospital death from cardiovascular disease where death usually occurs within the hour of emergence of symptoms. This thesis uses proteomics-based methods to investigate and validate a protein biomarker that could stratify patients at risk of SCD needing an intra-cardiac defibrillator implantation. Proteomics is the study of the whole proteome which are sets of proteins that are derived from biological fluids and tissue, these proteins could potentially be used as biomarkers to detect whether patients are at risk of SCD and if they would benefit of receiving an implantation of an intra-cardiac defibrillator (ICD).

Patient samples were acquired from the ULTIMATE patient cohort from the biobank at the Glenfield hospital. These samples were used to validate and quantify the 5 candidate biomarkers; and were also used in the shotgun metabolomic part of the study.

A metabolomic study was also conducted using, two different columns in positive ionization, C18 and hydrophilic interactions chromatography (HILIC); metabolomics is the study of small molecules called metabolites which are classed as end products of biological processes. These metabolites were investigated to see if there are any relationships between the 5 candidate biomarkers Apolipoprotein-L1 (APOL1), Cell Division Control Protein 42 Homolog (CDC42), Thrombospondin-1 (TSP1), Prolyl-3 hydroxylase-3 (P3h3), and Hyaluronan binding protein-2 (HABP2) and any signaling pathways. The 5 candidate biomarkers were initially verified using the Waters Synapt G2S HDMSE travelling wave ion mobility spectrometry (TWIMS) and time of flight (TOF) technologies with label free protein quantification along with multiple reaction monitoring (MRM) and verification using Water UPLC coupled with the Xevo nano-Acquity triple quadrupole mass spectrometer electrospray ionization. Shotgun metabolomics was carried out using the Waters G2 Synapt mass spectrometry coupled to liquid chromatography (LC), this provided qualitative and quantitative data. Shotgun metabolomic data identified compounds of interest which were related to cardiomyopathies and could be investigated further for potential biomarkers for identifying patients at risk of SCD.This thesis employs one of the candidate biomarkers Prolyl-3 hydroxylase-3 (P3h3), to be used as a surrogate for an isotopically labelled internal standard, this was found to be more cost effective and a sustainable way of uses animal plasma waste. Results from the validation study positively validated one of the 5 protein-based biomarkers hyaluronan binding protein-2 (HABP2) within the ULTIMATE patient cohort, the next step would be to validate this biomarker in the larger MINERVA patient cohort before being considered for clinical use. The European rabbit (Oryctolagus Cuniculus) P3H3 was identified to replace the human P3H3 as a surrogate as the peptide sequences were distinguishable but almost homologous.