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Proteomic analysis of HDL isolates reveals method-driven variability between ultracentrifugation and immunoaffinity chromatography: an interlaboratory approach

ABSTRACT: Therefore, the aim of our study is to systematically compare the effect of two different methods of HDL isolation, namely UC and IAC, performed in two independent research laboratories. For this purpose, we used the same plasma pool and protocols optimized to evaluate the influence of those variables in HDL proteome composition.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Francielle Aguiar Gomes

LAB HEAD: Graziella Eliza Ronsein

PROVIDER: PXD066558 | Pride | 2026-01-23

REPOSITORIES: Pride

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			Action	DRS
	241116_HDL_100ng_DIA_HolzerLab_IAC_Day1_A.raw	Raw
	241116_HDL_100ng_DIA_HolzerLab_IAC_Day1_B.raw	Raw
	241116_HDL_100ng_DIA_HolzerLab_IAC_Day1_C.raw	Raw
	241116_HDL_100ng_DIA_HolzerLab_IAC_Day2_A.raw	Raw
	241116_HDL_100ng_DIA_HolzerLab_IAC_Day2_B.raw	Raw

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Proteomic analysis of HDL isolates reveals method-driven variability: An interlaboratory approach.

Gomes Francielle Aguiar FA Souza Junior Douglas Ricardo DR Holzer Michael M Ronsein Graziella Eliza GE

Journal of lipid research 20251205 1

The high-density lipoprotein (HDL) is the most heterogeneous and protein-rich lipoprotein, and its complex proteome has been correlated with many distinct properties. However, the lack of a standardized approach for HDL isolation, combined with the fact that different methods capture overlapping, but distinct HDL subspecies make the establishment of a composition-function relationship a challenge. Key factors influencing HDL proteomic profile are the isolation methodology and the technical varia ...[more]

PMID: 41354322

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Proteomic dynamic changes in HDL subfractions from mouse plasma

Project description:HDL proteome dynamics may determine HDL cardioprotective functions. We aimed to characterize proteome and lipid profiles in small, medium and large (S/M/L) HDL fractions and analyze their functions. We characterized mouse HDL fractions by their high phospholipid (PL) content and collected S/M/L-HDLsubfractions by using fast protein liquid chromatography. The pooled S/L/M-HDL elution was subjected to mass spectrometry (MS) analysis using a Qstar XL-MS system, performing HDL subpopulation proteomic analysis. Fifty-one HDL proteins (39 in S-HDL, 27 in M-HDL and 29 in L-HDL) were identified and grouped into 4 functional categories (5 in lipid metabolism, 24 in immune response, 7 in coagulation, and 14 others). There were 16, 3 and 7 proteins present in only the S-HDL, M-HDL and L-HDL subfractions, respectively, and 11 proteins overlapped in all S/M/L-HDL subfractions. The dynamic changes in relative HDL protein levels were then characterized based on their functional groups by biological and bioinformatical methods.

2019-11-12 | PXD011185 | Pride

HDL Isolated by Immunoaffinity, Ultracentrifugation, or Precipitation is Compositionally and Functionally Distinct.

Project description:The HDL proteome has been widely recognized as an important mediator of HDL function. While a variety of HDL isolation methods exist, their impact on the HDL proteome and its associated function remain largely unknown. Here, we compared three of the most common methods for HDL isolation, namely immunoaffinity (IA), density gradient ultracentrifugation (UC), and dextran-sulfate precipitation (DS), in terms of their effects on the HDL proteome and associated functionalities. We used state-of-the-art mass spectrometry to identify 171 proteins across all three isolation methods. IA-HDL contained higher levels of paraoxonase 1, apoB, clusterin, vitronectin, and fibronectin, while UC-HDL had higher levels of apoA2, apoC3, and α-1-antytrypsin. DS-HDL was enriched with apoA4 and complement proteins, while the apoA2 content was very low. Importantly, size-exclusion chromatography analysis showed that IA-HDL isolates contained subspecies in the size range above 12 nm, which were entirely absent in UC-HDL and DS-HDL isolates. Analysis of these subspecies indicated that they primarily consisted of apoA1, IGκC, apoC1, and clusterin. Functional analysis revealed that paraoxonase 1 activity was almost completely lost in IA-HDL, despite high paraoxonase content. We observed that the elution conditions, using 3M thiocyanate, during IA resulted in an almost complete loss of paraoxonase 1 activity. Notably, the cholesterol efflux capacity of UC-HDL and DS-HDL was significantly higher compared to IA-HDL. Together, our data clearly demonstrate that the isolation procedure has a substantial impact on the composition, subclass distribution, and functionality of HDL. In summary, our data show that the isolation procedure has a significant impact on the composition, subclass distribution and functionality of HDL. Our data can be helpful in the comparison, replication and analysis of proteomic datasets of HDL.

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Project description:High-density lipoproteins (HDL) are nanoparticles with >80 associated proteins, phospholipids, cholesterol and cholesteryl esters. We have identified and quantified the ultracentrifugation isolated HDL proteome across 93 strains of mice, a diverse inbred strains of mice, Hybrid Mouse Diversity Panel (HMDP).

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Apolipoprotein A1 forms 5/5 and 5/4 antiparallel dimers in human high-density lipoprotein

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Structure dynamics of ApoA-I amyloidogenic variants in small HDL increase their ability to mediate cholesterol efflux

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2020-11-20 | PXD021266 | Pride

sRNA sequencing of HDL from human donors

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Molecular mechanisms vascular smooth muscle cell (VSMC) acquisition of macrophage features in response to cholesterol loading

Project description:In this study, murine primary aortic smooth muscle cells (SMCs) were transcriptionally profiled at baseline, after 3 d of cholesterol loading, and after 3 d of subsequent cholesterol unloading with HDL treatment, to identify vascular SMC genes that are transcripionally dysregulated in response to cholesterol loading and/or unloading. Mouse aortic SMCs were isolated from thoracic aortas of 8 week-old C57BL/6 mice (as described in Rong et al., Proc Nat Acad Sci USA, v100, pp13531M-bM-^@M-^S13536, 2003) and subconfluent cell preparations were harvested and transcriptionally profiled (baseline group) or treated with 10 M-NM-<g/mL cholesterol:methyl-M-NM-2-cyclodextrin complex (1:6 molar ratio) for 72 h for cholesterol loading. Of the cholesterol-loaded cells, samples were transcriptionally profiled (cholesterol samples) or treated for an additional 72 h with 100 M-NM-<g/mL human HDL and then transcriptionally profiled (HDL treatment). For all transcriptional profiling, RNA was isolated from cells using TRIzol, and labeled aRNA was prepared for hybridization to Affymetrix Mouse Genome 430 2.0 microarrays according to the manufacturer's protocol. Probe-level intensities were background-adjusted and normalized (in two separate analyses, one with baseline and cholesterol-treated samples, and the other analysis with all three sample groups) using Robust Multichip Average and combined into probesets using probe-to-probeset mappings from the University of Michigan CustomCDF project based on Ensembl Gene identifiers (release 59). Using the data from the analysis that combined the baseline and cholesterol-loaded samples, log2 probeset intensities were compared the between baseline and cholesterol sample groups using a one-way ANOVA with a P value cutoff determined by a Benjamini-Hochberg false discovery rate threshold of 0.05. Analysis (I): baseline and cholesterol-treated sample groups. Analysis (II): all three sample groups.

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Project description:Background: High density lipoprotein (HDL) protects the artery wall by removing cholesterol from lipid-laden macrophages. However, recent evidence suggests that it might also inhibit atherogenesis by combating inflammation. Methods and Results: To identify potential anti-inflammatory mechanisms, we challenged macrophages with lipopolysaccharide (LPS), an inflammatory microbial ligand for Toll-like receptor 4 (TLR4). HDL inhibited the expression of 33% (301 of 911) of the genes normally induced by LPS, microarray analysis revealed. One of its major targets was the type I interferon response pathway, a family of potent viral immunoregulators controlled by TLR4 and the TRAM/TRIF signaling pathway. Unexpectedly, HDL’s ability to inhibit gene expression was independent of cellular cholesterol stores. Moreover, it was unaffected by downregulation of two ATP-binding cassette transporters, ABCA1 and ABCG1, that promote cholesterol efflux. To examine the pathway’s potential in vivo relevance, we used mice deficient in apolipoprotein (apo) A-I, HDL’s major protein. After infection with Salmonella (a Gram-negative bacterium that expresses LPS), apoA-I–deficient mice had 6-fold higher plasma levels of interferon-beta-a key regulator of the type I interferon response than did wild-type mice. Conclusions: HDL inhibits a subset of LPS-stimulated macrophage genes that regulate the type I interferon response, and its action is independent of sterol metabolism. These findings raise the possibility that regulation of macrophage genes by HDL might link innate immunity and cardioprotection. 12 arrays, 3 experimental groups, mMncN (no treatment), mMncL (LPS treated, exposed for 4 h to serum-free medium or serum-free medium supplemented with 100 ng/mL of LPS), and mMwtL (HDL treated, Macrophages were treated for 4 h with serum-free medium or serum-free medium supplemented with 50 mg/mL of HDL, washed twice with PBS).

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