Advertisement

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

Open AccessPublished:October 29, 2022DOI:https://doi.org/10.1016/j.jlr.2022.100307

      Abstract

      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.

      Graphical Abstract

      Supplementary key words

      Abbreviations:

      AMBP (adipocyte plasma membrane-associated protein), DIA (data independent acquisition), DS (dextran-sulfate precipitation), IA (immunoaffinity), HGR (haptoglobin-related protein), IGHG1 (immunoglobulin gamma-1 chain C region), IGκC (immunoglobulin kappa chain C region), ITIH4 (Inter-alpha-trypsin inhibitor heavy chain H4), PLTP (Phospholipid transfer protein), PYCOX1 (Prenylcysteine oxidase 1), UC (ultracentrifugation)
      Since the discovery of HDL, a multitude of methods have been developed to isolate HDL (
      • Jones H.B.
      • Gofman J.W.
      • Lindgren F.T.
      • Lyon T.P.
      • Graham D.M.
      • Strisower B.
      • et al.
      Lipoproteins in atherosclerosis.
      ). Ultracentrifugation (UC) is still the most commonly used method to isolate HDL since it was introduced by Havel et al. several decades ago (
      • Havel R.J.
      • Eder H.A.
      • Bragdon J.H.
      The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum.
      ). Many different versions of UC methods have been developed, and the differences are related to centrifugation time, centrifugation force, chemicals used to adjust plasma density, osmotic pressure present, and whether or not a density gradient was used. However, HDL isolation by UC is a lengthy procedure that is less suitable for clinical use. As a result, precipitation methods have been developed for the selective removal of lipoproteins from serum. These methods can also be used to isolate intact HDL particles and commonly use polyanions such as dextran sulfate (
      • Burstein M.
      • Scholnick H.R.
      • Morfin R.
      Rapid method for the isolation of lipoproteins from human serum by precipitation with polyanions.
      ). Precipitation of lipoproteins by dextran sulfate in the presence of divalent cations is depended on both the positive charge and negative charge of the lipoproteins protein moiety as well as the charged groups of the phospholipids present. (
      • Nishida T.
      • Cogan U.
      Nature of the interaction of dextran sulfate with low density lipoproteins of plasma.
      ) The interaction forms insoluble complexes that precipitate and can be solubilized again by the removal the reagents. In recent years, isolation of HDL by immunoaffinity (IA) chromatography using specific antibodies for apoA1 has gained importance and represents an alternative method that reflects Alaupovic's apolipoprotein-based definition of lipoprotein classes. (
      • Alaupovic P.
      The concept of apolipoprotein-defined lipoprotein families and its clinical significance.
      ,
      • Kostner G.
      • Alaupovic P.
      Studies of the composition and structure of plasma lipoproteins. C- and N-terminal amino acids of the two nonidentical polypeptides of human plasma apolipoprotein A.
      ) Recently, Furtado et al. used IA chromatography to define 16 unique HDL subspecies based on the presence or absence of specific proteins besides apoA1 (
      • Furtado J.D.
      • Yamamoto R.
      • Melchior J.T.
      • Andraski A.B.
      • Gamez-Guerrero M.
      • Mulcahy P.
      • et al.
      Distinct proteomic signatures in 16 HDL (High-Density Lipoprotein) subspecies.
      ). IA chromatography has several advantages over UC, as it does not require centrifugal forces and high osmotic pressure and can isolate apoA1-containing particles from the entire size/density range of HDL. While these advantages are promising, the method also has drawbacks, such as rather low yield, high cost, and concerns about the specificity of the antibodies used. Over the past decade proteomics has expanded the list of HDL-associated proteins to over 200, while lipidomics has provided further new insights into the complexity of HDL-associated lipids (
      • Davidson W.S.
      • Shah A.S.
      • Sexmith H.
      • Gordon S.M.
      The HDL proteome watch: compilation of studies leads to new insights on HDL function.
      ,
      • Vaisar T.
      • Pennathur S.
      • Green P.S.
      • Gharib S.A.
      • Hoofnagle A.N.
      • Cheung M.C.
      • et al.
      Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL.
      ,
      • Kontush A.
      • Lhomme M.
      • Chapman M.J.
      Unravelling the complexities of the HDL lipidome.
      ,
      • Davidson W.S.
      • Silva R.A.
      • Chantepie S.
      • Lagor W.R.
      • Chapman M.J.
      • Kontush A.
      Proteomic analysis of defined HDL subpopulations reveals particle-specific protein clusters: relevance to antioxidative function.
      ,
      • Holzer M.
      • Birner-Gruenberger R.
      • Stojakovic T.
      • El-Gamal D.
      • Binder V.
      • Wadsack C.
      • et al.
      Uremia alters HDL composition and function.
      ).
      A key factor in the quantification of the HDL proteome besides a precise quantification method is the isolation methodology (
      • Ronsein G.E.
      • Vaisar T.
      Deepening our understanding of HDL proteome.
      ). Although much effort has been invested into the development of these protocols, there is little understanding of the impact of different isolation techniques on HDL composition and function. The aim of our study was to systematically compare composition and function HDL particles isolated by common methods, such as IA, UC, and dextran-sulfate precipitation (DS). For that purpose, we used a combination of untargeted (data-dependent acquisition, DDA) and targeted (data-independent acquisition, DIA) mass spectrometry methods for precise proteomic analysis of isolated HDLs and assessed key metrics of HDL function, such as cholesterol efflux capacity, paraoxonase 1 activity, and endothelial barrier promoting activity.

      Materials and Methods

      Blood collection

      Blood was sampled from 18 healthy control subjects (inclusion criteria: apparently healthy, free of chronic disease, and currently not on medication). Three independent serum pools of six participants each, matched for age and sex, were prepared from the sera of the 18 subjects (see Table 1). All subjects signed an informed consent form in agreement with the Institutional Review Board of the Medical University of Graz. All methods were carried out in accordance with the approved by the local ethics committee (Nr.: 21–523 ex 09/10) and the principles of the Declaration of Helsinki.

      HDL isolation

      UC

      Serum density was adjusted with potassium bromide (Sigma, Vienna, Austria) to 1.24 g/ml, and a two-step density gradient was generated in centrifuge tubes (16 × 76 mm, Beckman, Nr. 342,413) by layering 3 ml density-adjusted plasma (1.24 g/ml) underneath a KBr-density solution (1.063 g/ml) as described (
      • Sattler W.
      • Mohr D.
      • Stocker R.
      Rapid isolation of lipoproteins and assessment of their peroxidation by high-performance liquid chromatography postcolumn chemiluminescence.
      ). Tubes were sealed and centrifuged at 65.000 rpm (415.000 g) for 6 h in a 90Ti fixed angle rotor (Beckman Instruments, Krefeld, Germany). After centrifugation, the HDL-containing band was collected, desalted via PD10 columns (GE Healthcare, Vienna, Austria) and either immediately used for experiments or stored with 5% sucrose at −70°C (
      • Holzer M.
      • Kern S.
      • Trieb M.
      • Trakaki A.
      • Marsche G.
      HDL structure and function is profoundly affected when stored frozen in the absence of cryoprotectants.
      ).

      IA purification of HDL subspecies

      HDL subspecies were isolated from human sera of healthy volunteers with modifications as described (
      • Furtado J.D.
      • Yamamoto R.
      • Melchior J.T.
      • Andraski A.B.
      • Gamez-Guerrero M.
      • Mulcahy P.
      • et al.
      Distinct proteomic signatures in 16 HDL (High-Density Lipoprotein) subspecies.
      ,
      • Burnap S.A.
      • Sattler K.
      • Pechlaner R.
      • Duregotti E.
      • Lu R.
      • Theofilatos K.
      • et al.
      PCSK9 activity is potentiated through HDL binding.
      ). Serum was incubated overnight with gentle turning at 4°C with Sepharose 4B resin coupled to a polyclonal anti–apoA-I antibody (Catalog # S81-104, Fortis life science) at a ratio of 0.25 ml serum per 1 ml antibody resin. The unbound fraction was removed by washing three times with PBS. For elution, apoA1-resin was incubated for 5 min with 3 M sodium thiocyanate, and the eluate collected. Elution was repeated for a total of three times. Eluted samples were concentrated on Vivaspin Turbo 15 columns (VWR, Germany), followed by buffer exchanged to PBS on PD MiniTrap G-10 columns (Cytiva Life Science).

      DS

      We used a commercial available kit from Cell Biolabs (Nr.: STA-607). The isolation was performed according to the manufacturer’s instructions.

      Size-exclusion chromatography

      NGC QUEST FPLC System (Bio-Rad, Germany) equipped with a Superdex 200 Increase 50/300 column (Nr.: 28,990,944, Cytiva Life Science) was used with DPBS containing 0.9 mM CaCl2 and 0.49 mM MgCl2, pH 7.4 as running buffer (Nr.: 14,040,133, ThermoFisher, Germany). HDL samples (0.5 mg protein) were loaded with a 0.25 ml loop and were separated with a constant flow of 0.5 ml/min. For HDL2/3 separations, 0.25 ml fraction was collected, and fractions pooled as indicated in Fig. 2.

      Gel electrophoresis and blotting

      For native gel electrophoresis, isolated HDL (5–15 μg protein per lane) was separated by native gel electrophoresis on 4%–16% gels (BN1004BOX, ThermoFisher). Gels were run at constant voltage of 150 V for 120 min. As a high molecular weight marker (NativeMark, Nr.: LC0725, Life Technologies, Austria), containing bovine serum albumin (7.1 nm), lactate dehydrogenase (8.2 nm), B-phycoerythrin (10.5 nm, apoferritin band 1 (12.2 nm), and apoferritin band 2 (18.0 nm) was used to estimate the size of HDL. Afterwards, gels were either stained with a freshly prepared solution of Coomassie Brilliant Blue G-250 overnight (ThermoFisher) or used for blotting. Gels were transferred to polyvinylidene difluoride membranes with an iBlot 2 Dry Blotting System at 100V for 7 min at RT. Membranes were probed blocked with 5 % milk in PBS for 1 hour and incubated with the following primary antibodies diluted in 5 % milk in PBS overnight at 4°C. The list of antibodies used can be found in the supporting information (supplemental Table S1).
      Membranes were washed carefully for at least three times with wash buffer and incubated with secondary HRP-conjugated antibodies (goat anti-rabbit, Nr.:111-005-045; goat anti-mouse, Nr.:115-005-146; rabbit anti-goat, Nr.:305-005-045) for 2 h at RT. Membranes were carefully washed and developed using Clarity ECL western reagents (Nr.: 170–5061, Bio-Rad, Austria). Detection was performed on a Chemidoc Touch imaging system (Bio-Rad, Austria).

      Proteomics

      HDL digestion

      HDL (5–10 μg) was solubilized in 100 mM ammonium bicarbonate in the presence of 0.2% sodium deoxycholate (Sigma-Aldrich), reduced with 5 mM dithiothreitol (Bio-Rad) and alkylated with iodoacetamide (Bio-Rad). Proteins were digested with trypsin from Promega (1:40, w:w, enzyme: HDL protein) for 4 h at 37°C. A second trypsin aliquot was added to the samples (1:50, w:w HDL protein) and incubated overnight at 37°C (
      • Silva A.R.M.
      • Toyoshima M.T.K.
      • Passarelli M.
      • Di Mascio P.
      • Ronsein G.E.
      Comparing data-independent acquisition and parallel reaction monitoring in their abilities to differentiate high-density lipoprotein subclasses.
      ). Digestion was stopped, and sodium deoxycholate was precipitated with 0.6% trifluoroacetic acid (Sigma-Aldrich). Samples were desalted according to the StageTip protocol (
      • Rappsilber J.
      • Mann M.
      • Ishihama Y.
      Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips.
      ), dried under vacuum and stored at −80ºC until MS analyses. Before MS analyses, samples were resuspended in 0.1% formic acid (Fluka), with a final protein concentration of 50 ng/μl.

      MS proteomic analyses

      Digested HDL proteins (50 ng) were loaded onto a trap column (nanoViper C18, 3 μm, 75 μm × 2 cm, Thermo Scientific) and eluted onto a C18 column (nanoViper, 2 μm, 75 μm × 15 cm, Thermo Scientific). Peptides were analyzed using an Easy-nLC 1200 UHPLC system (Thermo Scientific) coupled to an Orbitrap Fusion Lumos (Thermo Scientific) equipped with a nanospray FlexNG ion source (Thermo Scientific) in a 44 min gradient and normalized collision energy of 30 for HCD fragmentation. For untargeted analysis (DDA), peptides were analyzed using MS1 resolution of 120,000 (at m/z 200) with AGC target set to 4 × 105, m/z range of 350–1550, and maximum injection time of 50 ms. MS2 resolution was set at 30,000 (at m/z 200) with AGC target of 5 × 104 and maximum injection time of 54 ms. For targeted analysis (DIA), peptides were quantified using Orbitrap resolution of 30,000 (at m/z 200) with AGC target of 5 × 105, precursor m/z range of 400–900, scan range of product ions between m/z 100 and 1000, maximum injection time of 54 ms, and isolation windows of 25 m/z with 0.5 m/z margins.

      MS data processing

      MaxQuant software (version 2.0.1.0) was used to search raw shotgun MS spectra against the human proteome (Uniprot, 20,371 entries). The criteria for protein detection and quantification included at least two peptides (at least one of them unique), with methionine oxidation and protein N-terminal acetylation selected as variable modifications and carbamidomethylation of cysteine as fixed modification. DDA data were used to build a library for DIA analyses. DIA data were analyzed using Skyline software (version 21.1.0.146) as described (
      • Silva A.R.M.
      • Toyoshima M.T.K.
      • Passarelli M.
      • Di Mascio P.
      • Ronsein G.E.
      Comparing data-independent acquisition and parallel reaction monitoring in their abilities to differentiate high-density lipoprotein subclasses.
      ). For DIA, we used only unique peptides (at least 4 transitions per peptide) and avoided choosing peptides susceptible to ex vivo modification (i.e., containing methionine), peptides with high interference signals and mass error higher than 10 ppm. All peaks used for quantification were manually inspected to select the best transitions and ensure correct peak detection and integration. A pooled quality control (QC) was made by combining unfractionated digested HDL samples isolated by IA, UC, and DS (n = 3 each). This QC was injected 7 times intercalating with samples to control for technical variability. Proteins and peptides that achieved coefficients of variation lower than 15% in the pooled HDL QC were considered for quantification. For each protein, quantification was performed by summing up the areas of 2–6 most intense peptides. To give an estimate of protein abundance within each HDL, the value obtained for each protein quantification was divided by the theoretical number of tryptic peptides.

      Arylesterase activity of paraoxonase-1

      Ca2+-dependent arylesterase activity was determined with a photometric assay using phenylacetate as previously described. HDL (0.5 μg protein) was added to 200 μl buffer containing 100 mmol/L Tris, 2 mmol/L CaCl2 (pH 8.0), and 1 mmol/L phenylacetate to a 96-well quartz glass plate (Hellma, Baden, Germany). The rate of hydrolysis of phenylacetate was monitored by the increase of absorbance at 270 nm, and readings were taken every 15 s at room temperature to generate a kinetic plot. The slope from the kinetic chart was used to determine the increase in fluorescence per minute. Enzymatic activity was calculated with the Beer-Lambert Law from the molar extinction coefficient of 1310 mol1∗L−1∗cm−1 for phenylacetate.

      Cholesterol efflux assay

      J774.2 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) in the presence of 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were plated on 48-well plates (300,000 cells/well), cultured for 24 h, and loaded with 0.5 μCi/ml radiolabeled [3H]-cholesterol in DMEM supplemented with 2% fetal bovine serum and 1% penicillin/streptomycin in the presence or absence of 0.3 mM 8-(4-chlorophenylthio)-cyclic adenosine monophosphate overnight to induce the expression of adenosine triphosphate-binding cassette subfamily A member 1 (ABCA1). After labeling, cells were rinsed with serum-free DMEM containing 1% penicillin/streptomycin and equilibrated with serum-free DMEM containing penicillin/streptomycin and 2 mg/ml bovine serum albumin for 2 h. Subsequently [3H]-cholesterol efflux was determined by incubating cells for 3 h with 50 μg protein/ml HDL. Cholesterol efflux was expressed as radioactivity in the cell culture supernatant relative to total radioactivity (in the cell culture supernatant and cells) of three independent experiments respectively, measured in duplicates. All steps were performed in the presence of 2 μg/ml of an acyl coenzyme A cholesterol acyltransferase inhibitor (Sandoz 58-035).

      Endothelial barrier promoting activity assay

      96W20idf chips (Ibidi, Germany) were incubated with 10 mM L-cysteine for 10 min at RT, washed twice with PBS, followed by a coating with 1% gelatine for 30 min at 37°C. The human umbilical vein cell line (Ea.hy926) was maintained in DMEM containing 10% FBS, 1% HAT and 1% Penstrept, seeded at 30.000 per well, and grown to full confluence for two days. Cells were serum-starved prior to experiments and baseline recorded until stable, which was routinely achieved after 1–2 h. Afterward, 100 μg/ml HDL was added, and impedance monitored over a period of 20 h. For quantification, we used the impedance recorded at 4000 Hz at the 10 h time point.

      Statistical analysis

      Differences between two groups (Fig. 4C, D; with and without thiocyanate treatment) were analyzed with the Student’s t test. Differences between three groups (IA vs. UC vs. DS) were analyzed with one-way ANOVA followed by Bonferroni’s multiple comparison test. Significance was accepted at ∗P < 0.05 and ∗∗P < 0.01. Statistical analyses were performed with GraphPad Prism, Version 6, and SPSS, Version 26. Principal component analysis was performed on Perseus (v.2.0.3.1) with imputation of missing values (less than 2% of the total).

      Results

      We isolated HDL with three different methods, being IA, UC, and DS (Table 1) from three independent serum pools. While UC is based on the density difference of HDL to other serum compounds, IA directly targets the major HDL associated protein apoA1. Precipitation uses a specific interaction of polyanions with charged groups on proteins and lipids to precipitate HDL. The baseline characteristics for the serum pools used are given in the supplemental Table S2.
      Table 1Overview of methods used to isolate HDL
      AcronymMethodMode of SeparationCompounds UsedFactors to be ConsideredProteins Detected by MS
      IA-HDLImmunoaffinityapoA-I contentapoA-I antibody
      • -
        Antibody specificity
      142
      UC-HDLUltracentrifugationdensityPotassium bromide
      • -
        Osmotic pressure
      • -
        Centrifugal force
      112
      DS-HDLPrecipitationsolubilityDextran sulfate
      • -
        Precipitation specificity
      123
      DS, dextran-sulfate precipitation; IA, immunoaffinity; UC, ultracentrifugation.
      To investigate the differences in the proteome of isolated HDL, we used a combination of untargeted DDA and targeted DIA mass spectrometry methods. Global protein discovery by DDA allowed us to detect a total of 171 proteins across all three isolation methods, with 142 proteins being present in IA-HDL, 112 proteins in UC-HDL, and 123 proteins in DS-HDL (Table 1 and supplemental data). We used the DDA results to construct a library to analyze DIA samples. DIA chromatograms obtained in the Skyline software were carefully analyzed, and peptides of 51 HDL proteins that were consistently and confidently detected in a pool of all samples with low coefficient of variation (<15%) were selected for label-free relative quantitative analysis. Raw data and calculations of the mass spectrometry data are provided in the Supplementary Data section.
      The results from the targeted DIA proteomics clearly showed that the composition of HDL from the different isolation methods is distinct (Fig. 1A). A principal component analysis of the results showed a distinct cluster for each individual isolation method indicating that the methods are indeed different (Fig. 1B). The principal component analysis clusters further suggested that the tested methods are reproducible, which was also confirmed by gel electrophoresis analysis of individual isolations from the same serum pool (supplemental Fig. S1). The results indicate that the major HDL-associated protein, apoA-I, accounts for 53.4%–59.8% of the total isolated protein and was lowest in UC-HDL (Table 2). Further changes in UC-HDL where higher levels of apoA2, apoC3, and α-1-antytrypsin, while IA-HDL had higher levels of paraoxonase 1, PLTP, apoB, clusterin, vitronectin, fibronectin as well as a series of complement proteins (Table 2). DS-HDL was markedly enriched in apoA4 and complement proteins, while the content of apoA2, apoD, and paraoxonase 1 was very low compared to the other methods (Table 2).
      Figure thumbnail gr1
      Fig. 1HDL isolated by different methods is compositional distinct. A: Heatmap of proteomics DIA data from HDL isolation by immunoaffinity (IA-HDL), ultracentrifugation (UC-HDL), or dextran-sulfate precipitation (DS-HDL) from three independent samples. B: Principal component analysis of HDL and HDL2/3. For each protein, the integrated area of the DIA analysis obtained in Skyline software was used to build the heat map and the PCA. Proteins with CV <15% in the pooled HDL quality control (n = 51) were included in PCA analysis. DIA, data-independent acquisition.
      Table 2Mass spectrometry analysis of HDL
      Protein NameImmunoaffinityUltracentrifugationPrecipitation
      % of Total Protein (Mean ± Stdev)% of Total Protein (Mean ± Stdev)% of Total Protein (Mean ± Stdev)
      Apolipoprotein A156,8413 ± 24,20953,4370 ± 18,33759,7746 ± 14,581
      UC-HDL versus DS-HDL; P < 0.05.
      Apolipoprotein A221,9839 ± 14,24025,2083 ± 06,84118,0152 ± 08,703
      IA-HDL versus UC-HDL.
      ,
      IA-HDL versus DS-HDL.
      ,
      UC-HDL versus DS-HDL; P < 0.05.
      Apolipoprotein C152,658 ± 07,13468,114 ± 08,13079,474 ± 07,789
      IA-HDL versus DS-HDL.
      Apolipoprotein C324,509 ± 06,71843,525 ± 07,86833,411 ± 04,694
      IA-HDL versus UC-HDL.
      Apolipoprotein C225,120 ± 05,52231,688 ± 07,10831,557 ± 07,099
      Apolipoprotein D21,532 ± 02,49522,346 ± 03,65104,058 ± 00,523
      IA-HDL versus DS-HDL.
      ,
      UC-HDL versus DS-HDL; P < 0.05.
      Serum amyloid A413,113 ± 01,88318,542 ± 01,00023,306 ± 02,227
      IA-HDL versus UC-HDL.
      ,
      IA-HDL versus DS-HDL.
      Albumin07,869 ± 04,38109,337 ± 02,05607,463 ± 00,513
      Paraoxonase 109,036 ± 00,35206,791 ± 01,52201,989 ± 00,144
      IA-HDL versus DS-HDL.
      ,
      UC-HDL versus DS-HDL; P < 0.05.
      Apolipoprotein M02,774 ± 00,23602,914 ± 00,51401,807 ± 00,236
      IA-HDL versus DS-HDL.
      ,
      UC-HDL versus DS-HDL; P < 0.05.
      Apolipoprotein E04,830 ± 00,49602,817 ± 00,47603,636 ± 00,735
      IA-HDL versus UC-HDL.
      Platelet basic protein00,148 ± 00,09701,489 ± 00,38800,546 ± 00,030
      IA-HDL versus UC-HDL.
      ,
      UC-HDL versus DS-HDL; P < 0.05.
      Apolipoprotein A404,266 ± 02,29101,245 ± 00,09826,678 ± 02,132
      IA-HDL versus DS-HDL.
      ,
      UC-HDL versus DS-HDL; P < 0.05.
      Apolipoprotein F00,533 ± 00,07800,797 ± 00,06600,280 ± 00,030
      IA-HDL versus UC-HDL.
      ,
      UC-HDL versus DS-HDL; P < 0.05.
      α-1-antitrypsin00,340 ± 00,07200,687 ± 00,20100,302 ± 00,089
      IA-HDL versus UC-HDL.
      ,
      IA-HDL versus DS-HDL.
      ,
      UC-HDL versus DS-HDL; P < 0.05.
      Transthyretin00,641 ± 00,06300,547 ± 00,28300,147 ± 00,058
      IA-HDL versus DS-HDL.
      IGκC17,060 ± 04,75200,487 ± 00,01100,735 ± 00,063
      Apolipoprotein C400,290 ± 00,07000,379 ± 00,11000,186 ± 00,046
      Fetuin A00,140 ± 00,04200,346 ± 00,16900,841 ± 00,230
      IA-HDL versus DS-HDL.
      ,
      UC-HDL versus DS-HDL; P < 0.05.
      Apolipoprotein L100,390 ± 00,08200,257 ± 00,10100,157 ± 00,070
      IA-HDL versus DS-HDL.
      IGHG102,317 ± 00,87200,201 ± 00,05200,261 ± 00,039
      IA-HDL versus UC-HDL.
      ,
      IA-HDL versus DS-HDL.
      Thrombin00,978 ± 00,38200,175 ± 00,02800,287 ± 00,048
      IA-HDL versus UC-HDL.
      ,
      IA-HDL versus DS-HDL.
      Apolipoprotein H00,123 ± 00,06200,138 ± 00,05200,071 ± 00,013
      IA-HDL versus UC-HDL.
      ,
      IA-HDL versus DS-HDL.
      Clusterin09,980 ± 02,97100,131 ± 00,01801,032 ± 00,225
      IA-HDL versus UC-HDL.
      ,
      IA-HDL versus DS-HDL.
      Serotransferrin00,228 ± 00,12600,130 ± 00,01700,166 ± 00,007
      PLTP00,267 ± 00,02000,094 ± 00,00600,053 ± 00,004
      IA-HDL versus UC-HDL.
      ,
      IA-HDL versus DS-HDL.
      ,
      UC-HDL versus DS-HDL; P < 0.05.
      LCAT00,029 ± 00,00400,052 ± 00,01800,015 ± 00,004
      UC-HDL versus DS-HDL; P < 0.05.
      Complement C300,256 ± 00,07600,052 ± 00,01900,763 ± 00,157
      IA-HDL versus DS-HDL.
      ,
      UC-HDL versus DS-HDL; P < 0.05.
      Vitronectin02,077 ± 00,37200,049 ± 00,02301,818 ± 00,894
      IA-HDL versus UC-HDL.
      ,
      UC-HDL versus DS-HDL; P < 0.05.
      HGR00,427 ± 00,03800,038 ± 00,00800,028 ± 00,005
      IA-HDL versus UC-HDL.
      ,
      IA-HDL versus DS-HDL.
      PYCOX100,039 ± 00,01000,033 ± 00,00300,035 ± 00,006
      Protein AMBP00,025 ± 00,00600,032 ± 00,00900,033 ± 00,004
      Haptoglobin00,153 ± 00,02600,024 ± 00,00700,042 ± 00,022
      IA-HDL versus UC-HDL.
      ,
      IA-HDL versus DS-HDL.
      Angiotensinogen00,024 ± 00,00800,016 ± 00,01000,005 ± 00,003
      Apolipoprotein B00,150 ± 00,05900,014 ± 00,00600,002 ± 00,001
      IA-HDL versus UC-HDL.
      ,
      IA-HDL versus DS-HDL.
      α-2-macroglobulin00,101 ± 00,06400,010 ± 00,01300,032 ± 00,012
      Serum amyloid P-C00,778 ± 00,43700,008 ± 00,00800,053 ± 00,039
      IA-HDL versus UC-HDL.
      ,
      IA-HDL versus DS-HDL.
      Plasminogen00,050 ± 00,01900,007 ± 00,00200,474 ± 00,150
      IA-HDL versus DS-HDL.
      ,
      UC-HDL versus DS-HDL; P < 0.05.
      Complement C1qB01,505 ± 00,36500,005 ± 00,00500,010 ± 00,012
      IA-HDL versus UC-HDL.
      ,
      IA-HDL versus DS-HDL.
      CD5 antigen-like00,297 ± 00,03300,004 ± 00,00200,011 ± 00,004
      IA-HDL versus UC-HDL.
      ,
      IA-HDL versus DS-HDL.
      Complement C1qC02,089 ± 00,33100,003 ± 00,00600,007 ± 00,011
      IA-HDL versus UC-HDL.
      ,
      IA-HDL versus DS-HDL.
      Complement C1s01,719 ± 00,44800,003 ± 00,00300,018 ± 00,005
      IA-HDL versus UC-HDL.
      ,
      IA-HDL versus DS-HDL.
      Complement factor B00,004 ± 00,00200,003 ± 00,00200,045 ± 00,008
      IA-HDL versus UC-HDL.
      ,
      IA-HDL versus DS-HDL.
      Complement C1qA01,770 ± 00,17700,003 ± 00,00500,009 ± 00,013
      IA-HDL versus UC-HDL.
      ,
      IA-HDL versus DS-HDL.
      C4b-binding protein α00,045 ± 00,00300,003 ± 00,00500,036 ± 00,038
      Apolipoprotein(a)00,005 ± 00,00400,003 ± 00,00200,009 ± 00,001
      ITIH400,041 ± 00,01500,002 ± 00,00100,027 ± 00,002
      IA-HDL versus UC-HDL.
      ,
      UC-HDL versus DS-HDL; P < 0.05.
      Afamin00,003 ± 00,00100,001 ± 00,00000,007 ± 00,003
      UC-HDL versus DS-HDL; P < 0.05.
      Fibronectin00,434 ± 00,15100,001 ± 00,00100,130 ± 00,073
      IA-HDL versus UC-HDL.
      ,
      IA-HDL versus DS-HDL.
      Complement C1r00,581 ± 00,14700,001 ± 00,00100,008 ± 00,004
      IA-HDL versus UC-HDL.
      ,
      IA-HDL versus DS-HDL.
      Complement C500,004 ± 00,00100,000 ± 00,00000,045 ± 00,018
      IA-HDL versus DS-HDL.
      ,
      UC-HDL versus DS-HDL; P < 0.05.
      AMBP, adipocyte plasma membrane-associated protein; DS, dextran-sulfate precipitation; IA, immunoaffinity; HGR, haptoglobin-related protein; IGHG1, immunoglobulin gamma-1 chain C region; IGκC, immunoglobulin kappa chain C region; ITIH4, Inter-alpha-trypsin inhibitor heavy chain H4; PLTP, Phospholipid transfer protein; PYCOX1, Prenylcysteine oxidase 1; UC, ultracentrifugation.
      a IA-HDL versus UC-HDL.
      b IA-HDL versus DS-HDL.
      c UC-HDL versus DS-HDL; P < 0.05.
      To investigate the size distribution of isolated HDL, we used size-exclusion chromatography (SEC). Interestingly, we found that HDL isolated by IA contained a fraction larger than 12 nm, which was almost entirely absent with the other isolation methods. The distribution between the HDL2/3 subclasses was similar between IA-HDL and UC-HDL, while the HDL3 subclass was largely missing in DS-HDL (Fig. 2). To further investigate the different subclasses, we collected three major fraction (above 12 nm, between 12 – 7.5 nm and below 7.5 nm) from the SEC for proteomics analysis (Fig. 2).
      Mass spectrometry data indicated that the subclass larger than 12 nm, which was only present in IA-HDL, contained primarily apoA1 (24%), IGκC (13%), clusterin (10%), apoC1 (9%), and apoC2 (4%) together with a small amount of apoB (0.3%) (Table 3). The fraction larger than 12 nm from IA-HDL contained a large amount of various complement C1q proteins. However, recent reports have shown that this might be an artefact, at least in part, of protein isolation using sepharose as a matrix (
      • Roeth P.J.
      • Easterbrook-Smith S.B.
      C1q is a nucleotide binding protein and is responsible for the ability of clusterin preparations to promote immune complex formation.
      ). The size fraction above 12 nm was barely present in UC-HDL and DS-HDL, where it was mainly composed of apoA1 and apoA2 (Tables 4 and 5). The main HDL subclasses HDL2/3 is located in the size range between 7.5 and 12 nm (Fig. 2).
      Table 3Proteome of IA-HDL separated by size-exclusion chromatography
      Protein Name>12 nm12–7.5 nm<7.5 nm
      % of Total Protein (Mean ± Stdev)% of Total Protein (Mean ± Stdev)% of Total Protein (Mean ± Stdev)
      Apolipoprotein A124,4566 ± 55,80259,2482 ± 13,36574,2260 ± 28,776
      Apolipoprotein A273,363 ± 14,84025,6114 ± 20,96142,585 ± 16,097
      Apolipoprotein C191,468 ± 21,44045,224 ± 02,18401,224 ± 00,056
      Apolipoprotein C240,451 ± 21,39811,736 ± 01,84402,017 ± 02,278
      Apolipoprotein C324,340 ± 07,28208,126 ± 03,08004,682 ± 05,433
      Apolipoprotein D30,039 ± 07,60523,492 ± 01,69808,884 ± 01,352
      Serum amyloid A409,561 ± 01,55315,816 ± 00,80800,964 ± 00,422
      Paraoxonase 123,312 ± 03,60506,756 ± 00,42825,129 ± 06,302
      Apolipoprotein M02,951 ± 00,76603,200 ± 00,31700,310 ± 00,108
      Apolipoprotein E32,294 ± 13,02602,116 ± 00,25303,157 ± 01,612
      Apolipoprotein F00,416 ± 00,08600,428 ± 00,16400,552 ± 00,198
      Albumin05,457 ± 04,15401,937 ± 01,03010,2662 ± 56,717
      IGκC13,2735 ± 25,83711,183 ± 04,23804,061 ± 01,473
      Apolipoprotein A406,085 ± 02,70601,205 ± 00,83339,967 ± 21,512
      Apolipoprotein C400,981 ± 00,24200,198 ± 00,04200,001 ± 00,000
      Apolipoprotein L100,345 ± 00,06900,449 ± 00,07400,074 ± 00,044
      IGHG112,362 ± 03,60102,274 ± 00,71501,810 ± 00,670
      Transthyretin01,119 ± 00,17100,338 ± 00,07400,651 ± 00,101
      Clusterin96,794 ± 09,96007,937 ± 01,79204,788 ± 01,786
      α-1-antitrypsin00,346 ± 00,14700,154 ± 00,03902,789 ± 00,227
      PLTP01,554 ± 00,15600,247 ± 00,02900,029 ± 00,008
      LCAT00,059 ± 00,01400,017 ± 00,00500,176 ± 00,037
      PYCOX100,142 ± 00,06400,032 ± 00,00700,197 ± 00,090
      HGR02,184 ± 00,24200,445 ± 00,01700,190 ± 00,058
      Complement C301,734 ± 00,39200,238 ± 00,07700,583 ± 00,393
      Apolipoprotein H00,153 ± 00,21100,051 ± 00,05300,630 ± 00,241
      Vitronectin16,399 ± 03,97201,439 ± 00,20001,551 ± 01,089
      Haptoglobin00,955 ± 00,37300,172 ± 00,01900,083 ± 00,055
      Serotransferrin00,122 ± 00,13900,046 ± 00,05103,643 ± 02,490
      Thrombin05,376 ± 01,30900,736 ± 00,35400,813 ± 00,606
      Fetuin A00,095 ± 00,02400,021 ± 00,01200,859 ± 00,259
      Apolipoprotein B02,721 ± 00,62100,039 ± 00,02100,220 ± 00,159
      α-2-macroglobulin00,936 ± 00,27300,092 ± 00,06500,016 ± 00,019
      Protein AMBP00,071 ± 00,01800,011 ± 00,00100,050 ± 00,025
      Complement C1s12,312 ± 05,91601,748 ± 00,30300,589 ± 00,426
      Complement C1qB28,943 ± 05,12400,607 ± 00,12400,062 ± 00,029
      Angiotensinogen00,087 ± 00,04200,027 ± 00,00900,041 ± 00,011
      ITIH400,111 ± 00,06300,030 ± 00,00500,388 ± 00,258
      Complement C1qA36,711 ± 11,22000,665 ± 00,09200,057 ± 00,025
      Complement C1qC43,306 ± 08,05500,742 ± 00,14800,079 ± 00,014
      Complement C1r05,099 ± 02,05100,488 ± 00,06000,098 ± 00,040
      Fibronectin04,186 ± 02,86500,324 ± 00,02100,098 ± 00,027
      Serum amyloid P-C01,803 ± 01,03300,455 ± 00,38300,273 ± 00,265
      Afaminn.d.00,001 ± 00,00100,040 ± 00,015
      Apolipoprotein(a)00,079 ± 00,08900,001 ± 00,00100,075 ± 00,039
      CD5 antigen-like05,095 ± 01,45900,123 ± 00,04000,012 ± 00,005
      Plasminogen00,086 ± 00,07100,012 ± 00,00200,515 ± 00,241
      Platelet basic protein00,107 ± 00,07700,004 ± 00,00400,008 ± 00,011
      Complement factor B00,002 ± 00,00100,002 ± 00,00100,049 ± 00,022
      C4b-binding protein α00,507 ± 00,25900,017 ± 00,00300,006 ± 00,006
      Complement C500,079 ± 00,01600,003 ± 00,00200,001 ± 00,001
      AMBP, adipocyte plasma membrane-associated protein; IA, immunoaffinity; HGR, haptoglobin-related protein; IGHG1, immunoglobulin gamma-1 chain C region; IGκC, immunoglobulin kappa chain C region; ITIH4, Inter-alpha-trypsin inhibitor heavy chain H4; PLTP, Phospholipid transfer protein; PYCOX1, Prenylcysteine oxidase 1.
      Table 4Proteome of UC-HDL separated by size-exclusion chromatography
      Protein Name>12 nm12–7.5 nm<7.5 nm
      % of Total Protein (Mean ± Stdev)% of Total Protein (Mean ± Stdev)% of Total Protein (Mean ± Stdev)
      Apolipoprotein A138,3334 ± 64,50053,7352 ± 30,80058,8046 ± 65,767
      Apolipoprotein A224,1147 ± 73,58428,1440 ± 15,43679,256 ± 09,922
      Apolipoprotein C136,929 ± 28,50963,489 ± 07,78805,860 ± 01,115
      Apolipoprotein C226,747 ± 33,73228,274 ± 06,39007,655 ± 07,238
      Apolipoprotein C313,093 ± 13,28927,996 ± 09,24130,796 ± 31,871
      Apolipoprotein D71,473 ± 35,83326,704 ± 01,17006,499 ± 03,265
      Serum amyloid A408,685 ± 03,79118,430 ± 01,95203,737 ± 00,504
      Paraoxonase 115,327 ± 02,40407,212 ± 01,19601,309 ± 00,485
      Apolipoprotein M02,384 ± 01,93403,074 ± 00,34000,281 ± 00,045
      Apolipoprotein E26,266 ± 17,06202,245 ± 00,44000,674 ± 00,534
      Apolipoprotein F00,273 ± 00,08000,723 ± 00,11300,261 ± 00,240
      Albumin33,372 ± 24,59500,700 ± 00,59623,7596 ± 46,127
      IGκC42,290 ± 17,29700,421 ± 00,03300,143 ± 00,018
      Apolipoprotein A402,379 ± 02,67200,340 ± 00,20016,658 ± 01,531
      Apolipoprotein C400,507 ± 00,51200,336 ± 00,09700,004 ± 00,004
      Apolipoprotein L100,997 ± 00,94000,268 ± 00,12300,039 ± 00,029
      IGHG109,090 ± 05,04400,226 ± 00,06700,273 ± 00,022
      Transthyretin00,644 ± 00,63800,125 ± 00,06200,541 ± 00,331
      Clusterin12,400 ± 06,30600,124 ± 00,03500,089 ± 00,065
      α-1-antitrypsin00,918 ± 00,86000,100 ± 00,02710,523 ± 04,252
      PLTP07,338 ± 02,58300,092 ± 00,01000,005 ± 00,003
      LCAT00,012 ± 00,01000,053 ± 00,02100,051 ± 00,037
      PYCOX100,252 ± 00,22000,041 ± 00,00600,004 ± 00,005
      HGR00,659 ± 00,63100,040 ± 00,01300,002 ± 00,002
      Complement C301,417 ± 00,51700,037 ± 00,00800,536 ± 00,607
      Apolipoprotein H00,534 ± 00,70700,033 ± 00,02801,085 ± 00,343
      Vitronectin28,742 ± 44,07400,032 ± 00,02200,210 ± 00,109
      Haptoglobin01,804 ± 01,64300,027 ± 00,01000,004 ± 00,002
      Serotransferrin00,910 ± 00,91300,013 ± 00,01703,651 ± 00,614
      Thrombin00,333 ± 00,28600,010 ± 00,00400,035 ± 00,036
      Fetuin A00,818 ± 00,71200,009 ± 00,00603,504 ± 01,975
      Apolipoprotein B02,761 ± 01,91700,006 ± 00,00300,027 ± 00,019
      α-2-macroglobulin08,869 ± 08,83000,005 ± 00,00700,006 ± 00,007
      Protein AMBP01,613 ± 01,17400,005 ± 00,00200,107 ± 00,066
      Complement C1s00,916 ± 00,60800,005 ± 00,00300,001 ± 00,001
      Complement C1qB01,245 ± 01,26100,003 ± 00,00400,002 ± 00,004
      Angiotensinogen00,108 ± 00,09500,002 ± 00,00100,251 ± 00,198
      ITIH400,094 ± 00,09600,002 ± 00,00100,010 ± 00,008
      Complement C1qA01,156 ± 01,77500,001 ± 00,00200,001 ± 00,002
      Complement C1qC01,450 ± 02,11100,001 ± 00,002<00,001 ± 00,001
      Complement C1r00,231 ± 00,25500,001 ± 00,001<00,001 ± 00,001
      Fibronectin00,998 ± 01,36700,001 ± 00,00100,003 ± 00,005
      Serum amyloid P-C00,054 ± 00,09300,001 ± 00,001n.d.
      Afamin00,003 ± 00,00400,001 ± 00,00000,019 ± 00,001
      Apolipoprotein(a)00,382 ± 00,148<00,001 ± 00,00100,013 ± 00,001
      CD5 antigen-like01,777 ± 01,363<00,001 ± 00,00100,001 ± 00,002
      Plasminogen04,267 ± 06,898<00,001 ± 00,00100,084 ± 00,022
      Platelet basic protein01,741 ± 01,37100,001 ± 00,00000,076 ± 00,132
      Complement factor B00,014 ± 00,01800,001 ± 00,00000,072 ± 00,022
      C4b-binding protein α00,847 ± 01,365<00,001 ± 00,000<00,001 ± 00,000
      Complement C500,402 ± 00,418n.d.<00,001 ± 00,000
      AMBP, adipocyte plasma membrane-associated protein; HGR, haptoglobin-related protein; IGHG1, immunoglobulin gamma-1 chain C region; IGκC, immunoglobulin kappa chain C region; ITIH4, Inter-alpha-trypsin inhibitor heavy chain H4; PLTP, Phospholipid transfer protein; PYCOX1, Prenylcysteine oxidase 1; UC, ultracentrifugation.
      Table 5Proteome of DS-HDL separated by size-exclusion chromatography
      Protein Name>12 nm12–7.5 nm<7.5 nm
      % of Total Protein (Mean ± Stdev)% of Total Protein (Mean ± Stdev)% of Total Protein (Mean ± Stdev)
      Apolipoprotein A136,8407 ± 44,81564,5180 ± 19,45647,7952 ± 13,4399
      Apolipoprotein A218,1915 ± 49,07621,2809 ± 31,81618,741 ± 05,450
      Apolipoprotein C137,787 ± 24,33463,439 ± 15,59802,116 ± 00,814
      Apolipoprotein C214,327 ± 17,03920,779 ± 04,68604,392 ± 02,996
      Apolipoprotein C306,476 ± 01,75214,033 ± 07,62314,621 ± 12,044
      Apolipoprotein D24,572 ± 16,86604,078 ± 00,40700,733 ± 00,216
      Serum amyloid A412,968 ± 09,92119,578 ± 02,70701,055 ± 00,187
      Paraoxonase 106,999 ± 01,02302,732 ± 00,15800,286 ± 00,120
      Apolipoprotein M01,562 ± 00,82202,168 ± 00,04500,079 ± 00,036
      Apolipoprotein E48,968 ± 30,63402,555 ± 00,48101,935 ± 00,959
      Apolipoprotein F00,126 ± 00,09800,210 ± 00,01100,227 ± 00,199
      Albumin07,952 ± 06,34900,782 ± 00,65915,5234 ± 52,565
      IGκC21,585 ± 16,43200,637 ± 00,07300,189 ± 00,016
      Apolipoprotein A417,907 ± 02,53406,583 ± 00,35428,7272 ± 95,170
      Apolipoprotein C400,478 ± 00,28300,158 ± 00,061n.d.
      Apolipoprotein L101,343 ± 00,81400,196 ± 00,09800,025 ± 00,022
      IGHG101,550 ± 01,26700,424 ± 00,04900,389 ± 00,133
      Transthyretin00,243 ± 00,24100,061 ± 00,03800,188 ± 00,122
      Clusterin10,318 ± 06,72900,868 ± 00,15602,296 ± 01,039
      α-1-antitrypsin00,450 ± 00,36800,078 ± 00,04503,073 ± 00,479
      PLTP02,622 ± 00,61800,045 ± 00,00300,002 ± 00,002
      LCAT00,008 ± 00,01300,007 ± 00,00000,046 ± 00,027
      PYCOX100,107 ± 00,10000,036 ± 00,00700,011 ± 00,011
      HGR00,489 ± 00,43400,024 ± 00,00600,003 ± 00,002
      Complement C304,385 ± 02,61600,710 ± 00,17501,123 ± 00,374
      Apolipoprotein H00,087 ± 00,12600,052 ± 00,00600,357 ± 00,146
      Vitronecti16,6531 ± 12,93001,153 ± 00,52001,451 ± 00,922
      Haptoglobin02,749 ± 02,39000,041 ± 00,02100,005 ± 00,001
      Serotransferrin00,100 ± 00,13200,016 ± 00,01803,864 ± 02,021
      Thrombin17,950 ± 16,04700,188 ± 00,02500,151 ± 00,007
      Fetuin A02,169 ± 02,34900,114 ± 00,07606,957 ± 03,425
      Apolipoprotein B00,202 ± 00,14000,001 ± 00,00000,010 ± 00,012
      α-2-macroglobulin11,761 ± 05,91800,026 ± 00,01800,010 ± 00,015
      Protein AMBP00,175 ± 00,18000,027 ± 00,00600,020 ± 00,009
      Complement C1s02,417 ± 01,18300,016 ± 00,00900,004 ± 00,002
      Complement C1qB00,167 ± 00,14500,002 ± 00,00300,006 ± 00,010
      Angiotensinogen00,032 ± 00,03000,001 ± 00,00100,034 ± 00,014
      ITIH400,445 ± 00,42400,021 ± 00,00300,215 ± 00,029
      Complement C1qA00,242 ± 00,17100,001 ± 00,00100,006 ± 00,009
      Complement C1qC00,206 ± 00,25700,001 ± 00,00200,010 ± 00,017
      Complement C1r00,218 ± 00,17900,005 ± 00,00300,005 ± 00,006
      Fibronectin15,449 ± 12,74200,079 ± 00,03600,074 ± 00,091
      Serum amyloid P-C00,184 ± 00,16300,004 ± 00,00200,003 ± 00,006
      Afamin00,006 ± 00,00900,002 ± 00,00100,089 ± 00,039
      Apolipoprotein(a)00,003 ± 00,003<00,001 ± 00,00001,411 ± 00,756
      CD5 antigen-like02,889 ± 01,60300,002 ± 00,00100,021 ± 00,010
      Plasminogen00,495 ± 00,55900,007 ± 00,00210,985 ± 03,093
      Platelet basic protein00,348 ± 00,13700,004 ± 00,00401,872 ± 00,129
      Complement factor B00,005 ± 00,00800,028 ± 00,00700,388 ± 00,104
      C4b-binding protein α00,291 ± 00,42300,002 ± 00,00200,005 ± 00,003
      Complement C501,334 ± 00,95800,036 ± 00,01500,059 ± 00,035
      AMBP, adipocyte plasma membrane-associated protein; DS, dextran-sulfate precipitation; HGR, haptoglobin-related protein; IGHG1, immunoglobulin gamma-1 chain C region; IGκC, immunoglobulin kappa chain C region; ITIH4, Inter-alpha-trypsin inhibitor heavy chain H4; PLTP, Phospholipid transfer protein; PYCOX1, Prenylcysteine oxidase 1.
      In comparison to total HDL, the proportion of apoA1 increased from 53.4%–59.8% to 53.7%–64.5% and for apoA2 increased from 18.0%–25.2% to 21.3%–28.1% in purified HDL2/3. The data from HDL2/3 suggests an increase in apoA2 and lower levels of apoA1 in UC-HDL (Table 5). Such an increase could be due to either a loss of apoA-I and thus an accumulation of apoA2, which is known to be less exchangeable than apoA1 (
      • Pownall H.J.
      • Ehnholm C.
      The unique role of apolipoprotein A-I in HDL remodeling and metabolism.
      ) or from differences in the HDL subclass distribution across the isolation methods, since apoA2 is known to be preferentially associated with HDL3. The latter is certainly the case for DS-HDL, which has a much lower content of the smaller HDL3 subclass (Fig. 2). Looking at the apoA2/apoA1 ratio between the isolation methods, we found that the ratio was higher in UC-HDL compared to IA-HDL and DS-HDL (supplemental Table S3). Such an increase could be due to either a loss of apoA-I and thus an accumulation of apoA2, which is known to be less exchangeable than apoA1 (
      • Pownall H.J.
      • Ehnholm C.
      The unique role of apolipoprotein A-I in HDL remodeling and metabolism.
      ) or from differences in the HDL subclass distribution across the isolation methods, since apoA2 is known to be preferentially associated with HDL3. The latter is certainly the case for DS-HDL, which has a much lower content of the smaller HDL3 subclass (Fig. 2). Notable differences between the isolation methods for HDL2/3 were a lower content of apoC’s in IA-HDL, while clusterin and PLTP were higher (Table 3). UC-HDL contained high levels of apoC3 (Table 4), while DS-HDL contained more apoA4 and low levels of apoD and paraoxonase when compared to the other methods (Table 5).
      Figure thumbnail gr2
      Fig. 2Size-exclusion chromatography of isolated HDL. Separation of IA-HDL(A), UC-HDL (B) and DS-HDL (C) was performed on an NGC Quest FPLC system equipped with a Superdex 200 column. Dashed lines represent the usual size range of the main HDL2/3 subclass. DS, dextran-sulfate precipitation; IA, immunoaffinity chromatography; UC, ultracentrifugation.
      Notably, the content of a number of proteins decreased upon HDL2/3 purification in the range between 70% and 99%. A sharp decrease in protein abundance was observed for albumin (-86%), apoA4 (-73%), α-1-antytrypsin (-71%), apoB (-63%), serotransferrin (-87%), fetuin A (-90%), platelet basic protein (-99%), apo(a) (-88%), afamin (-65%), plasminogen (-90%), and a series of complement proteins (-60%-80%). After SEC separation, the majority of these proteins were either identified in the SEC fraction above 12 nm (most complement proteins, apo(a), apoB, platelet basic protein, plasminogen) or in the SEC fraction below 7.5 nm (α-1-antytrypsin, apoA4, fetuin A, afamin, serotranferrin) (Table 3, Table 4, Table 5). Our data suggest that the majority of these proteins is not present on the HDL2/3 subclass and more likely to be associated with other HDL subspecies or are co-isolates/contaminants of the isolation procedure itself.
      To further investigate the distribution of the identified proteins over the entire HDL size range, we performed native gel electrophoresis followed by Western blotting and antibody detection. The dashed line in Fig. 3 shows the size range of HDL2/3 between ∼7.5 and 12 nm. As expected, the highest abundance of apoA1 was observed throughout the HDL2/3 size range, while lower levels were visible at 5–7.5 nm indicating the presence of pre-β HDL in the isolates (Fig. 3). ApoA1 distribution was fairly even across HDL2/3 in IA-HDL and UC-HDL, while lower levels of apoA1 were observed in the smaller HDL3 fraction in DS HDL. This result is consistent with proteomics data (Table 2) and SEC analysis (Fig. 2). Other apoproteins such as apoA2, C1, C2, C3, E, and L1 showed a clear distribution consistent with the results of previous studies (
      • Asztalos B.F.
      • Schaefer E.J.
      • Horvath K.V.
      • Yamashita S.
      • Miller M.
      • Franceschini G.
      • et al.
      Role of LCAT in HDL remodeling: investigation of LCAT deficiency states.
      ,
      • Samanovic M.
      • Molina-Portela M.P.
      • Chessler A.-D.C.
      • Burleigh B.A.
      • Raper J.
      Trypanosome lytic factor, an antimicrobial high-density lipoprotein, ameliorates Leishmania infection.
      ). Analysis of the apoA4 distribution showed the presence of several different subgroups. One fraction had a size of around ∼7 nm and probably resembled the poorly-lipidated forms of apoA4 (
      • Asztalos B.F.
      • Schaefer E.J.
      • Horvath K.V.
      • Yamashita S.
      • Miller M.
      • Franceschini G.
      • et al.
      Role of LCAT in HDL remodeling: investigation of LCAT deficiency states.
      ). This fraction was also removed by SEC and therefore explains the large decrease in apoA4 content after purification of HDL2/3 (Table 3). Two other apoA4 subgroups were present in the size range of 8 and 12 nm, respectively. Interestingly, these subgroups with sizes of 8 and 12 nm were not present in UC-HDL, suggesting that they either have different densities or are lost due to gravitational forces or osmotic pressure during UC. Clusterin was found mainly in IA-HDL with a distribution ranging from 7.5 up to 20 nm, suggesting that several subclasses of HDL carry clusterin. Paraoxonase 1 was present in HDL2/3 as well as in smaller particles around ∼6.5 nm. α-1-antitrypsin was present in HDL over the entire size range of HDL, with a significant accumulation at around 6.5 nm. Consistent with this observation, data from purification of total HDL into HDL2/3 showed a 70% reduction in α-1-antrypsin content (Table 1 vs. Table 2). This result suggests that 30% of the isolated α-1-antitrypsin was present in the size range of HDL2/3. Haptoglobin-related protein was detected in HDL from all three isolation methods in a size range of ∼10–13 nm. ApoA-I, apoL1, and haptoglobin-related protein together form the lipid-rich trypanosome lytic factor 1 (
      • Smith A.B.
      • Esko J.D.
      • Hajduk S.L.
      Killing of trypanosomes by the human haptoglobin-related protein.
      ), which is a part of the innate immune system responsible for the protection against African trypanosomes and Leishmania (
      • Samanovic M.
      • Molina-Portela M.P.
      • Chessler A.-D.C.
      • Burleigh B.A.
      • Raper J.
      Trypanosome lytic factor, an antimicrobial high-density lipoprotein, ameliorates Leishmania infection.
      ,
      • Smith A.B.
      • Esko J.D.
      • Hajduk S.L.
      Killing of trypanosomes by the human haptoglobin-related protein.
      ). Western blot analyses suggest the presence of lytic trypanosome factor 1 in all three isolation methods. Complement C3 was present in IA and DS-HDL, while only traces were detectable in UC-HDL. Complement C3 has a molecular weight of ∼185 kDa, which corresponds to a large proportion of the complement C3 present in DS-HDL. Interestingly, IA-HDL and DS-HDL had a proportion of complement C3 in the size range ∼11–18 nm, well above the molecular weight of 185 kDa, suggesting interaction with HDL or other proteins. Transthyretin and albumin were detected in HDL by all three methods, especially in the size range of large HDL2. According to the proteomic data, albumin content was highest in UC-HDL, with the majority detected in the SEC traction below 7.5 nm smaller pre-β-HDL size fraction (Table 4). Serum amyloid A was present in all three isolated HDLs, covering the entire size range of HDL2/3. Of note, DS-HDL also contained serum amyloid A in the size fraction below 7.5 nm. Significant amounts of plasminogen were found only in DS-HDL in the size range of HDL3, while only small amounts were present in IA-HDL (Table 2). Western blot analysis from native gel electrophoresis can provide valuable insights into the distribution of proteins across different size ranges. However, due to the different nature of the isolation methods, direct comparison of the protein abundances between the different isolation methods was not possible. We found that IA purification of HDL resulted in more intense signals on Western blots, regardless of the actual protein load. Coomassie blue staining of native gels loaded with an equal amount of protein clearly showed that protein levels in the major HDL subclasses HDL2/3 were highest in UC-HDL (supplemental Fig. S1). In contrast, Western blot analysis of the content of apoA1 in these samples gave the highest signal in IA-HDL (Figs. 2 and S2). Elution of the bound protein in IA purification is performed with 3 M thiocyanate, a strong chaotropic agent, which disrupts antigen-antibody binding and releases the bound protein (
      • Dandliker W.B.
      • Alonso R.
      • de Saussure V.A.
      • Kierszenbaum F.
      • Levison S.A.
      • Schapiro H.C.
      The effect of chaotropic ions on the dissociation of antigen-antibody complexes.
      ). Since thiocyanate is known to break hydrophobic, ionic, and hydrogen bonds, we hypothesize that the enhanced detection of proteins from IA-HDL is due to thiocyanate-induced accessibility of antibody binding sites.
      Figure thumbnail gr3
      Fig. 3Detection of HDL-associated proteins after native-gel electrophoresis. Isolated HDL from immunoaffinity (IA), ultracentrifugation (UC), and dextran-sulfate (DS) precipitation isolation were loaded on 4%–16% native gels and separated by native gel electrophoresis. Gels were blotted and probed with specific antibodies: apoA1 (A1); apoA2 (A2); apoC1 (C1); apoC2 (C2); apoC3 (C3); apoE (E); apoL1 (L1); apoA4 (A4); clusterin (CLUS); paraoxonase 1 (PON1), α-1-antitrypsin (A1AT); retinol-binding protein 4 (RBP4); haptoglobin-related protein (HGR); complement C3 (CO3); transthyretin (TT); human serum albumin (HSA); serum amyloid A (SAA); plasminogen (PLAS). Dashed lines represent the usual size range of the main HDL2/3 subclass.

      Impact of different isolation methods on the functionality of HDL

      Next, we investigated whether the compositional differences observed in isolated HDL are linked to altered functionality. Cholesterol efflux was measured with the widely used system using cyclic adenosine monophosphate–treated J774 macrophages (
      • de la Llera-Moya M.
      • Drazul-Schrader D.
      • Asztalos B.F.
      • Cuchel M.
      • Rader D.J.
      • Rothblat G.H.
      The ability to promote efflux via ABCA1 determines the capacity of serum specimens with similar high-density lipoprotein cholesterol to remove cholesterol from macrophages.
      ). Within this system cholesterol efflux is primarily mediated by apoA1 through ABCA1 (∼40%) and aqueous diffusion (∼50%) and to lower extant by SR-BI (∼10%) (
      • de la Llera-Moya M.
      • Drazul-Schrader D.
      • Asztalos B.F.
      • Cuchel M.
      • Rader D.J.
      • Rothblat G.H.
      The ability to promote efflux via ABCA1 determines the capacity of serum specimens with similar high-density lipoprotein cholesterol to remove cholesterol from macrophages.
      ,
      • Adorni M.P.
      • Zimetti F.
      • Billheimer J.T.
      • Wang N.
      • Rader D.J.
      • Phillips M.C.
      • et al.
      The roles of different pathways in the release of cholesterol from macrophages.
      ). Importantly, we found that IA-HDL was significantly less effective at promoting cholesterol efflux from macrophages (Fig. 4A). Our data did not show a significant difference in apoA1 that would explain the reduction in cholesterol efflux from IA-HDL (Table 2). However, taking the SEC data showing the distribution of HDL subspecies into account (Fig. 2), we suggest that the reduced content of HDL2/3 might be involved in the reduction of cholesterol efflux capability. Interestingly, DS-HDL was most potent in promoting cholesterol efflux. Our data suggest an increased content of apoA1 together with an increased content of larger HDL2 particles as the prime causes. Paraoxonase 1 is an atheroprotective enzyme that is mainly bound to HDL in the circulation (
      • Aviram M.
      • Rosenblat M.
      • Bisgaier C.L.
      • Newton R.S.
      • Primo-Parmo S.L.
      • Du B.N.L.
      Paraoxonase inhibits high-density lipoprotein oxidation and preserves its functions. A possible peroxidative role for paraoxonase.
      ,
      • Mackness M.I.
      • Arrol S.
      • Abbott C.
      • Durrington P.N.
      Protection of low-density lipoprotein against oxidative modification by high-density lipoprotein associated paraoxonase.
      ,
      • Bhattacharyya T.
      • Nicholls S.J.
      • Topol E.J.
      • Zhang R.
      • Yang X.
      • Schmitt D.
      • et al.
      Relationship of paraoxonase 1 (PON1) gene polymorphisms and functional activity with systemic oxidative stress and cardiovascular risk.
      ). We found that paraoxonase 1 activity was highest in UC-HDL, whereas activity was greatly reduced in DS-HDL and almost completely lost in IA-HDL (Fig. 4B). Importantly, the activity measurements were in striking contrast to the proteomics data, which indicated the highest level of paraoxonase 1 in IA-HDL (Table 2). We suspected that the elution conditions during IA purification may have caused the loss of function. To mimic the conditions during the elution process, we incubated UC-HDL with 3M thiocyanate and then removed it by gel filtration. This treatment resulted in an almost complete loss of paraoxonase-1 activity of UC-HDL (Fig. 4D). The reduced activity in DS-HDL was consistent with a lower paraoxonase-1 mass content (Table 2). We further tested whether thiocyanate also affected cholesterol efflux activity by treating UC-HDL with 3M thiocyanate. However, treatment of HDL with 3M-thiocyanate had no effect on cholesterol efflux capacity (Fig. 4C). We also analyzed the ability of HDL to promote endothelial integrity using an electrical impedance sensing system. We found that all three HDL isolates were able to improve endothelial barrier function by ∼10%. However, no significant differences were found between the three isolation methods, although UC-HDL tended to have a lower capacity (Fig. 3D).
      Figure thumbnail gr4
      Fig. 4Functional characterization of HDL isolated by immunoaffinity (IA), ultracentrifugation (UC), or dextran-sulfate precipitation (DS). A: cholesterol efflux was assessed by incubating cAMP-stimulated J774.2 macrophages with 50 μg protein/ml HDL for 3 h. Cholesterol efflux is expressed as the radioactivity in the medium relative to total radioactivity in medium and cells. B: Arylesterase activity of HDL-associated paraoxonase was measured by using phenylacetate as substrate. C, D: UC-HDL was preincubated with 3M thiocyanate for 30 min. Subsequently, cholesterol efflux and paraoxonase activity were measured as indicated above. E: Endothelial barrier promoting activity of HDL was measured using an electrical impedance sensing system (ECIS). Ea.Hy926 cells were grown to a confluent monolayer and incubated with 100 μg/ml HDL and the impedance of the endothelial monolayer monitored over time. After 10 h impedance values were taken for quantification. All experiments were performed at least three times.

      Discussion

      While a variety of HDL isolation methods exist, their impact on the HDL proteome and its associated function remain largely unknown. In this study, we systematically compared the composition and function of HDL particles isolated by common methods such as IA, UC, and DS. Especially IA chromatography with apoA1-specific antibodies has emerged as an alternative method for isolating HDL because UC alters the composition of lipoproteins (
      • Aviram M.
      Plasma lipoprotein separation by discontinuous density gradient ultracentrifugation in hyperlipoproteinemic patients.
      ,
      • Fainaru M.
      • Glangeaud M.C.
      • Eisenberg S.
      Radioimmunoassay of human high density lipoprotein apo-protein A-1.
      ). In contrast to UC and precipitation methods, IA purification isolates HDL across the full size spectrum of all apoA1-containing particles.
      In the present study, we observed that IA-HDL contained subtypes that were completely absent in UC-HDL and DS-HDL and that differed in composition from the major forms of HDL. This is consistent with previous reports showing that apoA1 can form HDL subclasses with higher size ranges (
      • Jenne D.E.
      • Lowin B.
      • Peitsch M.C.
      • Böttcher A.
      • Schmitz G.
      • Tschopp J.
      Clusterin (complement lysis inhibitor) forms a high density lipoprotein complex with apolipoprotein A-I in human plasma.
      ,
      • de Silva H.V.
      • Stuart W.D.
      • Duvic C.R.
      • Wetterau J.R.
      • Ray M.J.
      • Ferguson D.G.
      • et al.
      A 70-kDa apolipoprotein designated ApoJ is a marker for subclasses of human plasma high density lipoproteins.
      ). Further experiments will be necessary to distinguish whether these subclasses consist of one primary component or of a variety of different particles and how they function.
      An interesting observation of the present study was that IA-HDL was less potent in promoting cholesterol efflux and almost completely lost its paraoxonase activity. Remarkably, the prime mediator of cholesterol efflux, apoA1, was more abundant in IA-HDL than in UC-HDL. Therefore, other factors like changes in the subclass distribution and in the lipidation status might be responsible for the impaired cholesterol efflux capacity. For ABCA1-mediated efflux, the size and lipidation status of the cholesterol acceptor is of critical importance, and small and lipid poor forms of HDL are more effective acceptors (
      • Yancey P.G.
      • Bortnick A.E.
      • Kellner-Weibel G.
      • de la Llera-Moya M.
      • Phillips M.C.
      • Rothblat G.H.
      Importance of different pathways of cellular cholesterol efflux.
      ). We did not observe enrichment of small lipid-poor forms of HDL, such as pre-β HDL, in IA-HDL, so other factors seem more important. However, the differences in HDL subclass distribution were evident from SEC analysis. IA-HDL contained a fraction above 12 nm that made up about 20% of the overall protein mass. If this subclass were less efficient in promoting cholesterol efflux, an overall decrease would thus be plausible.
      A further important observation of our study was that DS precipitation yielded HDL distinct of UC-HDL and IA-HDL. This is line with a recent report demonstrating that precipitations reagents can have a significant impact on the compositions and size of isolated HDL (
      • Davidson W.S.
      • Heink A.
      • Sexmith H.
      • Melchior J.T.
      • Gordon S.M.
      • Kuklenyik Z.
      • et al.
      The effects of apolipoprotein B depletion on HDL subspecies composition and function.
      ). DS does not to affect the size distribution of HDL but alters the quantity of a subset of apolipoproteins (
      • Davidson W.S.
      • Heink A.
      • Sexmith H.
      • Melchior J.T.
      • Gordon S.M.
      • Kuklenyik Z.
      • et al.
      The effects of apolipoprotein B depletion on HDL subspecies composition and function.
      ). However, DS seems to be less damaging to HDL than the widely used polyethylene glycol treatment of serum, which leads to significant changes in the size and in the apolipoprotein distribution of HDL (
      • de la Llera-Moya M.
      • Drazul-Schrader D.
      • Asztalos B.F.
      • Cuchel M.
      • Rader D.J.
      • Rothblat G.H.
      The ability to promote efflux via ABCA1 determines the capacity of serum specimens with similar high-density lipoprotein cholesterol to remove cholesterol from macrophages.
      ).
      SEC has been used as an alternative approach for the isolation of HDL (
      • Collins L.A.
      • Mirza S.P.
      • Kissebah A.H.
      • Olivier M.
      Integrated approach for the comprehensive characterization of lipoproteins from human plasma using FPLC and nano-HPLC-tandem mass spectrometry.
      ,
      • Collins L.A.
      • Olivier M.
      Quantitative comparison of lipoprotein fractions derived from human plasma and serum by liquid chromatography-tandem mass spectrometry.
      ). While SEC is a method that allows purification of proteins in their native form, many plasma proteins have molecular weights in the same range as HDL, e.g., albumin dimer (∼135 kDa), IgG (150–180 kDa), and complement C3 (180 kDa) (
      • Ronsein G.E.
      • Vaisar T.
      Deepening our understanding of HDL proteome.
      ). In addition, many protein complexes overlap with the size distribution of HDL. Therefore, SEC alone is only able to enrich serum for HDL. To overcome this restrictions, SEC has to be combined with other methods to isolate HDL with high purity, for example with a lipid binding resin (
      • Gordon S.M.
      • Deng J.
      • Lu L.J.
      • Davidson W.S.
      Proteomic characterization of human plasma high density lipoprotein fractionated by gel filtration chromatography.
      ) or UC (
      • Holzer M.
      • Kern S.
      • Birner-Grunberger R.
      • Curcic S.
      • Heinemann A.
      • Marsche G.
      Refined purification strategy for reliable proteomic profiling of HDL2/3: impact on proteomic complexity.
      ,
      • Zheng J.J.
      • Agus J.K.
      • Hong B.V.
      • Tang X.
      • Rhodes C.H.
      • Houts H.E.
      • et al.
      Isolation of HDL by sequential flotation ultracentrifugation followed by size exclusion chromatography reveals size-based enrichment of HDL-associated proteins.
      ,
      • Vickers K.C.
      • Palmisano B.T.
      • Shoucri B.M.
      • Shamburek R.D.
      • Remaley A.T.
      MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins.
      ). Adding SEC after UC has the advantage that proteins that overlap with HDL in density, but are not HDL associated, are removed by size separation. The results of previous studies by others and us suggested that many of the detected proteins within UC-HDL isolates are not present in the HDL2/3 size range (
      • Holzer M.
      • Kern S.
      • Birner-Grunberger R.
      • Curcic S.
      • Heinemann A.
      • Marsche G.
      Refined purification strategy for reliable proteomic profiling of HDL2/3: impact on proteomic complexity.
      ,
      • Zheng J.J.
      • Agus J.K.
      • Hong B.V.
      • Tang X.
      • Rhodes C.H.
      • Houts H.E.
      • et al.
      Isolation of HDL by sequential flotation ultracentrifugation followed by size exclusion chromatography reveals size-based enrichment of HDL-associated proteins.
      ). These proteins are found exclusively in the smaller pre-β sized fractions (
      • Holzer M.
      • Kern S.
      • Birner-Grunberger R.
      • Curcic S.
      • Heinemann A.
      • Marsche G.
      Refined purification strategy for reliable proteomic profiling of HDL2/3: impact on proteomic complexity.
      ,
      • Zheng J.J.
      • Agus J.K.
      • Hong B.V.
      • Tang X.
      • Rhodes C.H.
      • Houts H.E.
      • et al.
      Isolation of HDL by sequential flotation ultracentrifugation followed by size exclusion chromatography reveals size-based enrichment of HDL-associated proteins.
      ) and cross-linking experiments revealed that no protein-proteins interaction with major HDL proteins are observed (
      • Holzer M.
      • Kern S.
      • Birner-Grunberger R.
      • Curcic S.
      • Heinemann A.
      • Marsche G.
      Refined purification strategy for reliable proteomic profiling of HDL2/3: impact on proteomic complexity.
      ). When using a lipid binding resin to purify lipoproteins after SEC, it must be considered that other non-HDL plasma proteins with lipid-binding affinity are co-isolated. Moreover, re-solubilization of bound proteins requires enzymatic digestion. Therefore, the isolated lipoproteins cannot be used to study functional properties.
      In conclusion, our data are the first to provide an in depth assessment of proteomic features of HDL isolated by UC, IA purification, and DS. We have demonstrated that the use of different isolation methods resulted in the isolation of HDL that was compositionally and functionally distinct. This is of particular importance as especially the use of IA purification gains widespread use for HDL isolation from clinical cohorts (
      • Furtado J.D.
      • Ruotolo G.
      • Nicholls S.J.
      • Dullea R.
      • Carvajal-Gonzalez S.
      • Sacks F.M.
      Pharmacological inhibition of CETP (Cholesteryl Ester Transfer Protein) increases HDL (High-Density Lipoprotein) that contains ApoC3 and other HDL subspecies associated with higher risk of coronary heart disease.
      ,
      • Aroner S.A.
      • Furtado J.D.
      • Sacks F.M.
      • Tsai M.Y.
      • Mukamal K.J.
      • McClelland R.L.
      • et al.
      Apolipoprotein C-III and its defined lipoprotein subspecies in relation to incident diabetes: the multi-ethnic study of atherosclerosis.
      ,
      • Sacks F.M.
      • Liang L.
      • Furtado J.D.
      • Cai T.
      • Davidson W.S.
      • He Z.
      • et al.
      Protein-defined subspecies of HDLs (High-Density Lipoproteins) and differential risk of coronary heart disease in 4 prospective studies.
      ,
      • Yamamoto R.
      • Jensen M.K.
      • Aroner S.
      • Furtado J.D.
      • Rosner B.
      • Hu F.B.
      • et al.
      HDL containing apolipoprotein C-III is associated with insulin sensitivity: a multicenter cohort study.
      ). Special attention must be taken when IA isolated HDL will be used for functional assays as the elution conditions with 3M thiocyanate can significantly alter its functional properties. Our data showing that separation and purification of HDL subclasses have a profound effect on HDL structure and function may help in the selection of the most appropriate isolation method for experimental purposes.

      Data Availability

      The data supporting this study are available in the article, the supplemental data, or available from the corresponding author upon reasonable request.

      Supplemental Data

      This article contains supplemental data.

      Conflict of Interest

      The authors declare that they have no conflict of interest.

      Author Contributions

      M. H. and G. M. conceptualization; M. H., S. H., H. S. and G. E. R. methodology; M. H., S. H., and G. E. R. formal analysis; M. H., S. H., D. R. S. J., J. T. S., A. R., G. E. R., and G. M. investigation; S. H., and G. M. resources; M. H., D. R. S. J., and G. E. R. data curation; M. H. writing–original draft; M. H., S. H., D. R. S. J., J. T. S., A. R., G. E. R., and G. M. writing–review & editing; M. H. visualization; M. H. and G. M. supervision; S. H., and G. M. funding acquisition.

      Funding and Additional Information

      This research was funded by BioTechMed-Graz , Young Researcher Groups (YRG) and the Austrian Science Fund (FWF), V-530 to S. L.-H., DOC 31-B26 and DOC 129 B to G. M. G. E. R. has received funding from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grants # 2013/07937-8 , and 2016/00696-3 ). For the purpose of open access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission.

      References

        • Jones H.B.
        • Gofman J.W.
        • Lindgren F.T.
        • Lyon T.P.
        • Graham D.M.
        • Strisower B.
        • et al.
        Lipoproteins in atherosclerosis.
        Am. J. Med. 1951; 11: 358-380
        • Havel R.J.
        • Eder H.A.
        • Bragdon J.H.
        The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum.
        J. Clin. Invest. 1955; 34: 1345-1353
        • Burstein M.
        • Scholnick H.R.
        • Morfin R.
        Rapid method for the isolation of lipoproteins from human serum by precipitation with polyanions.
        J. Lipid Res. 1970; 11: 583-595
        • Nishida T.
        • Cogan U.
        Nature of the interaction of dextran sulfate with low density lipoproteins of plasma.
        J. Biol. Chem. 1970; 245: 4689-4697
        • Alaupovic P.
        The concept of apolipoprotein-defined lipoprotein families and its clinical significance.
        Curr. Atheroscler. Rep. 2003; 5: 459-467
        • Kostner G.
        • Alaupovic P.
        Studies of the composition and structure of plasma lipoproteins. C- and N-terminal amino acids of the two nonidentical polypeptides of human plasma apolipoprotein A.
        FEBS Lett. 1971; 15: 320-324
        • Furtado J.D.
        • Yamamoto R.
        • Melchior J.T.
        • Andraski A.B.
        • Gamez-Guerrero M.
        • Mulcahy P.
        • et al.
        Distinct proteomic signatures in 16 HDL (High-Density Lipoprotein) subspecies.
        Arterioscler. Thromb. Vasc. Biol. 2018; 38: 2827-2842
        • Davidson W.S.
        • Shah A.S.
        • Sexmith H.
        • Gordon S.M.
        The HDL proteome watch: compilation of studies leads to new insights on HDL function.
        Biochim. Biophys. Acta Mol. Cell Biol. Lipids. 2021; 1867: 159072
        • Vaisar T.
        • Pennathur S.
        • Green P.S.
        • Gharib S.A.
        • Hoofnagle A.N.
        • Cheung M.C.
        • et al.
        Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL.
        J. Clin. Invest. 2007; 117: 746-756
        • Kontush A.
        • Lhomme M.
        • Chapman M.J.
        Unravelling the complexities of the HDL lipidome.
        J. Lipid Res. 2013; 54: 2950-2963
        • Davidson W.S.
        • Silva R.A.
        • Chantepie S.
        • Lagor W.R.
        • Chapman M.J.
        • Kontush A.
        Proteomic analysis of defined HDL subpopulations reveals particle-specific protein clusters: relevance to antioxidative function.
        Arterioscler. Thromb. Vasc. Biol. 2009; 29: 870-876
        • Holzer M.
        • Birner-Gruenberger R.
        • Stojakovic T.
        • El-Gamal D.
        • Binder V.
        • Wadsack C.
        • et al.
        Uremia alters HDL composition and function.
        J. Am. Soc. Nephrol. 2011; 22: 1631-1641
        • Ronsein G.E.
        • Vaisar T.
        Deepening our understanding of HDL proteome.
        Expert Rev. Proteomics. 2019; 16: 749-760
        • Sattler W.
        • Mohr D.
        • Stocker R.
        Rapid isolation of lipoproteins and assessment of their peroxidation by high-performance liquid chromatography postcolumn chemiluminescence.
        Met. Enzymol. 1994; 233: 469-489
        • Holzer M.
        • Kern S.
        • Trieb M.
        • Trakaki A.
        • Marsche G.
        HDL structure and function is profoundly affected when stored frozen in the absence of cryoprotectants.
        J. Lipid Res. 2017; 58: 2220-2228
        • Burnap S.A.
        • Sattler K.
        • Pechlaner R.
        • Duregotti E.
        • Lu R.
        • Theofilatos K.
        • et al.
        PCSK9 activity is potentiated through HDL binding.
        Circ. Res. 2021; 129: 1039-1053
        • Silva A.R.M.
        • Toyoshima M.T.K.
        • Passarelli M.
        • Di Mascio P.
        • Ronsein G.E.
        Comparing data-independent acquisition and parallel reaction monitoring in their abilities to differentiate high-density lipoprotein subclasses.
        J. Proteome Res. 2020; 19: 248-259
        • Rappsilber J.
        • Mann M.
        • Ishihama Y.
        Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips.
        Nat. Protoc. 2007; 2: 1896-1906
        • Roeth P.J.
        • Easterbrook-Smith S.B.
        C1q is a nucleotide binding protein and is responsible for the ability of clusterin preparations to promote immune complex formation.
        Biochim. Biophys. Acta. 1996; 1297: 159-166
        • Pownall H.J.
        • Ehnholm C.
        The unique role of apolipoprotein A-I in HDL remodeling and metabolism.
        Curr. Opin. Lipidol. 2006; 17: 209-213
        • Asztalos B.F.
        • Schaefer E.J.
        • Horvath K.V.
        • Yamashita S.
        • Miller M.
        • Franceschini G.
        • et al.
        Role of LCAT in HDL remodeling: investigation of LCAT deficiency states.
        J. Lipid Res. 2007; 48: 592-599
        • Samanovic M.
        • Molina-Portela M.P.
        • Chessler A.-D.C.
        • Burleigh B.A.
        • Raper J.
        Trypanosome lytic factor, an antimicrobial high-density lipoprotein, ameliorates Leishmania infection.
        PLoS Pathog. 2009; 5e1000276
        • Smith A.B.
        • Esko J.D.
        • Hajduk S.L.
        Killing of trypanosomes by the human haptoglobin-related protein.
        Science. 1995; 268: 284-286
        • Dandliker W.B.
        • Alonso R.
        • de Saussure V.A.
        • Kierszenbaum F.
        • Levison S.A.
        • Schapiro H.C.
        The effect of chaotropic ions on the dissociation of antigen-antibody complexes.
        Biochemistry. 1967; 6: 1460-1467
        • de la Llera-Moya M.
        • Drazul-Schrader D.
        • Asztalos B.F.
        • Cuchel M.
        • Rader D.J.
        • Rothblat G.H.
        The ability to promote efflux via ABCA1 determines the capacity of serum specimens with similar high-density lipoprotein cholesterol to remove cholesterol from macrophages.
        Arterioscler. Thromb. Vasc. Biol. 2010; 30: 796-801
        • Adorni M.P.
        • Zimetti F.
        • Billheimer J.T.
        • Wang N.
        • Rader D.J.
        • Phillips M.C.
        • et al.
        The roles of different pathways in the release of cholesterol from macrophages.
        J. Lipid Res. 2007; 48: 2453-2462
        • Aviram M.
        • Rosenblat M.
        • Bisgaier C.L.
        • Newton R.S.
        • Primo-Parmo S.L.
        • Du B.N.L.
        Paraoxonase inhibits high-density lipoprotein oxidation and preserves its functions. A possible peroxidative role for paraoxonase.
        J. Clin. Invest. 1998; 101: 1581-1590
        • Mackness M.I.
        • Arrol S.
        • Abbott C.
        • Durrington P.N.
        Protection of low-density lipoprotein against oxidative modification by high-density lipoprotein associated paraoxonase.
        Atherosclerosis. 1993; 104: 129-135
        • Bhattacharyya T.
        • Nicholls S.J.
        • Topol E.J.
        • Zhang R.
        • Yang X.
        • Schmitt D.
        • et al.
        Relationship of paraoxonase 1 (PON1) gene polymorphisms and functional activity with systemic oxidative stress and cardiovascular risk.
        JAMA. 2008; 299: 1265-1276
        • Aviram M.
        Plasma lipoprotein separation by discontinuous density gradient ultracentrifugation in hyperlipoproteinemic patients.
        Biochem. Med. 1983; 30: 111-118
        • Fainaru M.
        • Glangeaud M.C.
        • Eisenberg S.
        Radioimmunoassay of human high density lipoprotein apo-protein A-1.
        Biochim. Biophys. Acta. 1975; 386: 432-443
        • Jenne D.E.
        • Lowin B.
        • Peitsch M.C.
        • Böttcher A.
        • Schmitz G.
        • Tschopp J.
        Clusterin (complement lysis inhibitor) forms a high density lipoprotein complex with apolipoprotein A-I in human plasma.
        J. Biol. Chem. 1991; 266: 11030-11036
        • de Silva H.V.
        • Stuart W.D.
        • Duvic C.R.
        • Wetterau J.R.
        • Ray M.J.
        • Ferguson D.G.
        • et al.
        A 70-kDa apolipoprotein designated ApoJ is a marker for subclasses of human plasma high density lipoproteins.
        J. Biol. Chem. 1990; 265: 13240-13247
        • Yancey P.G.
        • Bortnick A.E.
        • Kellner-Weibel G.
        • de la Llera-Moya M.
        • Phillips M.C.
        • Rothblat G.H.
        Importance of different pathways of cellular cholesterol efflux.
        Arterioscler. Thromb. Vasc. Biol. 2003; 23: 712-719
        • Davidson W.S.
        • Heink A.
        • Sexmith H.
        • Melchior J.T.
        • Gordon S.M.
        • Kuklenyik Z.
        • et al.
        The effects of apolipoprotein B depletion on HDL subspecies composition and function.
        J. Lipid Res. 2016; 57: 674-686
        • Collins L.A.
        • Mirza S.P.
        • Kissebah A.H.
        • Olivier M.
        Integrated approach for the comprehensive characterization of lipoproteins from human plasma using FPLC and nano-HPLC-tandem mass spectrometry.
        Physiol. Genomics. 2010; 40: 208-215
        • Collins L.A.
        • Olivier M.
        Quantitative comparison of lipoprotein fractions derived from human plasma and serum by liquid chromatography-tandem mass spectrometry.
        Proteome Sci. 2010; 8: 42
        • Gordon S.M.
        • Deng J.
        • Lu L.J.
        • Davidson W.S.
        Proteomic characterization of human plasma high density lipoprotein fractionated by gel filtration chromatography.
        J. Proteome Res. 2010; 9: 5239-5249
        • Holzer M.
        • Kern S.
        • Birner-Grunberger R.
        • Curcic S.
        • Heinemann A.
        • Marsche G.
        Refined purification strategy for reliable proteomic profiling of HDL2/3: impact on proteomic complexity.
        Sci. Rep. 2016; 638533
        • Zheng J.J.
        • Agus J.K.
        • Hong B.V.
        • Tang X.
        • Rhodes C.H.
        • Houts H.E.
        • et al.
        Isolation of HDL by sequential flotation ultracentrifugation followed by size exclusion chromatography reveals size-based enrichment of HDL-associated proteins.
        Sci. Rep. 2021; 1116086
        • Vickers K.C.
        • Palmisano B.T.
        • Shoucri B.M.
        • Shamburek R.D.
        • Remaley A.T.
        MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins.
        Nat. Cell Biol. 2011; 13: 423-433
        • Furtado J.D.
        • Ruotolo G.
        • Nicholls S.J.
        • Dullea R.
        • Carvajal-Gonzalez S.
        • Sacks F.M.
        Pharmacological inhibition of CETP (Cholesteryl Ester Transfer Protein) increases HDL (High-Density Lipoprotein) that contains ApoC3 and other HDL subspecies associated with higher risk of coronary heart disease.
        Arterioscler. Thromb. Vasc. Biol. 2022; 42: 227-237
        • Aroner S.A.
        • Furtado J.D.
        • Sacks F.M.
        • Tsai M.Y.
        • Mukamal K.J.
        • McClelland R.L.
        • et al.
        Apolipoprotein C-III and its defined lipoprotein subspecies in relation to incident diabetes: the multi-ethnic study of atherosclerosis.
        Diabetologia. 2019; 62: 981-992
        • Sacks F.M.
        • Liang L.
        • Furtado J.D.
        • Cai T.
        • Davidson W.S.
        • He Z.
        • et al.
        Protein-defined subspecies of HDLs (High-Density Lipoproteins) and differential risk of coronary heart disease in 4 prospective studies.
        Arterioscler. Thromb. Vasc. Biol. 2020; 40: 2714-2727
        • Yamamoto R.
        • Jensen M.K.
        • Aroner S.
        • Furtado J.D.
        • Rosner B.
        • Hu F.B.
        • et al.
        HDL containing apolipoprotein C-III is associated with insulin sensitivity: a multicenter cohort study.
        J. Clin. Endocrinol. Metab. 2021; 106: e2928-e2940