Development of an absolute quantitation method of blood protein biomarkers for risk prediction of myocardial infarction events

Abstract

Myocardial infarction is the death of myocardial cells and the result of insufficient blood flow to the myocardial tissue. Caused by rupture of atherosclerotic plaque. The aim of the project was to develop an absolute quantitation method for 32 proteins in human plasma that are potential predictive blood protein biomarkers of myocardial infarction, which could be used in risk stratification. Additional objectives were to develop an automatic sample preparation method using a liquid handling robot, optimise protein digestion and perform absolute quantitation of individual plasma samples. Absolute quantitation was performed by using synthetically labelled and unlabelled proteotypic peptides of the 32 proteins as standards. A targeted absolute quantitation method using a bottom-up proteomics workflow was developed, implementing a liquid handling robot in sample preparation and nanoflow liquid chromatography using the Evosep One platform coupled to a triple quadrupole mass spectrometer. Collision energy optimisation of multiple reaction monitoring transitions and optimisation of the digestion process were performed, resulting in improved chromatographic peaks. The sample preparation was characterised, where peptide loss in SPE, peptide carry-over and peptide standard treatment were analysed. Absolute quantitation of individual plasma samples was successful for 12 of the 32 proteins analysed, which had good precision and passed the designated quality criteria of having at least six calibration standards and 66% of total quality controls and quality control per level within ± 20% of the theoretical concentration. Further investigation of method is required to quantitate all 32 proteins.