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Proteomic analysis of HDL isolates reveals method-driven variability: An interlaboratory approach.
Francielle Aguiar Gomes1, Douglas Ricardo Souza Junior1, Michael Holzer2
1Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, Brazil.
Journal of Lipid Research
|December 7, 2025
Summary
Methodological variability significantly impacts high-density lipoprotein (HDL) proteome analysis. Standardized isolation methods are crucial for reproducible HDL protein composition studies, though they may capture distinct HDL subspecies.
Area of Science:
- Lipidomics
- Proteomics
- Biochemistry
Background:
- High-density lipoprotein (HDL) is a heterogeneous, protein-rich particle with a complex proteome linked to various functions.
- Establishing a composition-function relationship for HDL is challenging due to diverse isolation methods yielding distinct subspecies.
- Methodological variability and interlaboratory technical differences in HDL isolation significantly influence proteomic profiles.
Purpose of the Study:
- To evaluate intralaboratory repeatability and intra- and interlaboratory reproducibility of two common HDL isolation methods: ultracentrifugation (UC) and immunoaffinity chromatography against APOA1 (IAC).
- To assess the influence of isolation methodology on HDL-associated protein composition and abundance.
- To determine the impact of interlaboratory technical variability on HDL proteome analysis.
Main Methods:
- Isolation of HDL particles using ultracentrifugation (UC) and immunoaffinity chromatography against APOA1 (IAC).
- Comprehensive evaluation of intralaboratory repeatability and intra- and interlaboratory reproducibility for both methods.
- Analysis of HDL-associated protein composition and abundance, focusing on the top 15 most abundant proteins.
Main Results:
- Methodological variability, not interlaboratory differences, explained nearly 60% of the variance in the HDL proteome.
- The top 15 HDL proteins constitute over 90% of HDL protein mass, irrespective of the isolation method.
- Protein quantification variability was inversely associated with protein abundance, with 11 (UC) and 10 (IAC) proteins showing <25% CV across interlaboratory reproducibility tests.
Conclusions:
- Methodological choices in HDL isolation profoundly impact proteomic outcomes.
- Standardized HDL isolation protocols can achieve acceptable reproducibility within and across laboratories.
- Different standardized methods may capture distinct HDL subspecies, highlighting the need for careful method selection based on research objectives.
