Purification and performance analysis of the LipoCAT-2 recombinant protein for the absolute quantification of apolipoproteins in biological samples

• Grant number: 25/05110-6
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: July 01, 2025
• End date: June 30, 2026
• Field of knowledge: Biological Sciences - Biochemistry
• Principal Investigator: Graziella Eliza Ronsein
• Grantee: Vinicius Diniz Nassin
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Known in clinical medicine as "good cholesterol," HDL (High-Density Lipoprotein) is composed of a diverse collection of particles that vary in density, function, and lipidic and proteic content. In this way, the current clinical measurement of HDL particles in circulation - their cholesterol content (HDL-C) - fails to capture their compositional and functional complexity. The HDL proteome varies in immune and cardiovascular diseases, and such alterations may serve as biomarkers for those. The accurate and precise protein quantification is a prerequisite for biomarker discovery and validation. However, the absolute quantification of HDL-associated proteins remains a challenge. To overcome this, we designed the recombinant protein LipoCAT-1, composed of peptides from the 53 most commonly found proteins in HDL particles, according to the literature. After evaluating its analytical performance, modifications were made to its structure, giving rise to the improved recombinant protein LipoCAT-2, which contains 48 peptides from proteins relevant to HDL, flanking sequences and two purification sites. In the current work, the protein will be expressed in E. coli, purified using Immobilized Metal Affinity Chromatography (IMAC), and the process of purification monitored via SDS-PAGE and protein concentration measurements using the Bradford assay. Subsequently, LipoCAT-2, expressed in labeled media (15N), will be used as an internal standard to the absolute quantification of HDL proteins, evaluating its analytical performance in both simple (isolated HDL) and complex (human plasma) biological samples. High-resolution mass spectrometry-based proteomics will be employed to monitor key analytical parameters such as limits of detection and quantification, linearity and others. This project has the potential to provide a method for the absolute quantification of HDL proteins in clinical samples, which may contribute to the identification of biomarkers and a better comprehension of HDL's function in a various pathological conditions.