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.