Reproducible Determination of High-Density Lipoprotein Proteotypes.

High-density lipoprotein (HDL) is a heterogeneous mixture of blood-circulating multimolecular particles containing many different proteins, lipids, and RNAs. Recent advancements in mass spectrometry-based proteotype analysis show promise for the analysis of proteoforms across large patient cohorts. In order to create the required spectral libraries enabling these data-independent acquisition (DIA) strategies, HDL was isolated from the plasma of more than 300 patients with a multiplicity of physiological HDL states. HDL proteome spectral libraries consisting of 296 protein groups and more than 786 peptidoforms were established, and the performance of the DIA strategy was benchmarked for the detection of HDL proteotype differences between healthy individuals and a cohort of patients suffering from diabetes mellitus type 2 and/or coronary heart disease. Bioinformatic interrogation of the data using the generated spectral libraries showed that the DIA approach enabled robust HDL proteotype determination. HDL peptidoform analysis enabled by using spectral libraries allowed for the identification of post-translational modifications, such as in APOA1, which could affect HDL functionality. From a technical point of view, data analysis further shows that protein and peptide quantities are currently more discriminative between different HDL proteotypes than peptidoforms without further enrichment. Together, DIA-based HDL proteotyping enables the robust digitization of HDL proteotypes as a basis for the analysis of larger clinical cohorts.