Development and optimization of a sensitive analytical method to quantify homocitrulline in lipoprotein fractions from plasma samples

Abstract

Lipoproteins may be modified by the post-translational modification of carbamylation, which alters the protein’s properties, leading to loss of function. Carbamylated lipoproteins are proven to acquire atherogenic properties, and this will increase the risk of cardiovascular diseases. Therefore, carbamylated-derived products, such as homocitrulline, can function as biomarkers for these diseases. The analytical method for homocitrulline quantification is currently technically demanding and time-consuming and has previously been proven not sensitive enough. Therefore, this project aims to develop and optimize methods for sample preparation and analysis to quantify homocitrulline in lipoprotein fractions in plasma samples from individuals with an increased risk of developing cardiovascular disease. This method optimization is essential, as sensitive analytical methods are required for homocitrulline to function as an important biomarker in individuals with an increased risk of cardiovascular disease. The low-density and high-density lipoprotein fractions were separated from other plasma components using sequential density ultracentrifugation and characterized by NanoDrop spectroscopy, and SDS-PAGE gel electrophoresis. To quantify homocitrulline in these fractions, the proteins were hydrolyzed into free amino acids by acid hydrolysis. Acid hydrolysis was performed in hydrolysis glass vials and a vacuum microreactor, and the hydrolysates were then dried using different numbers of evaporation steps, and with different sample volumes, to optimize the hydrolysis method. The dry hydrolysates were reconstituted in a solution suitable for analysis on a liquid chromatography/mass spectrometry instrument. The reconstitution solution was optimized by examining several different reconstitution solutions. Ultimately, the hydrolyzed and redissolved protein fractions were analyzed with hydrophilic interaction liquid chromatography coupled with time-of-flight mass spectrometry using electrospray ionization in positive mode. Several analysis parameters, such as mobile phase, gradient elution method, and extraction method, were examined to optimize the quantification of homocitrulline. These optimizations resulted in a functioning analysis method. Due to unexpected high loss of the internal standard in the quantitative experiment, the calibration curve generated to quantify homocitrulline could not correct for analyte loss and other variations during the analytical method, and may thus only be used to estimate concentrations, assuming that all samples behave similarly. This entire analytical method can, therefore, be further developed and optimized in order to measure accurate levels of homocitrulline in plasma samples, which is a prerequisite for homocitrulline to function as a valuable diagnostic and prognostic biomarker for cardiovascular diseases.