Isolation of HDL by sequential flotation ultracentrifugation followed by size exclusion chromatography reveals size-based enrichment of HDL-associated proteins.

High-density lipoprotein (HDL) particles have multiple beneficial and cardioprotective roles, yet our understanding of their full structural and functional repertoire is limited due to challenges in separating HDL particles from contaminating plasma proteins and other lipid-carrying particles that overlap HDL in size and/or density. Here we describe a method for isolating HDL particles using a combination of sequential flotation density ultracentrifugation and fast protein liquid chromatography with a size exclusion column. Purity was visualized by polyacrylamide gel electrophoresis and verified by proteomics, while size and structural integrity were confirmed by transmission electron microscopy. This HDL isolation method can be used to isolate a high yield of purified HDL from a low starting plasma volume for functional analyses. This method also enables investigators to select their specific HDL fraction of interest: from the least inclusive but highest purity HDL fraction eluting in the middle of the HDL peak, to pooling all of the fractions to capture the breadth of HDL particles in the original plasma sample. We show that certain proteins such as lecithin cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP), and clusterin (CLUS) are enriched in large HDL particles whereas proteins such as alpha-2HS-glycoprotein (A2HSG), alpha-1 antitrypsin (A1AT), and vitamin D binding protein (VDBP) are enriched or found exclusively in small HDL particles.

Effect of High-Density Lipoprotein from Healthy Subjects and Chronic Kidney Disease Patients on the CD14 Expression on Polymorphonuclear Leukocytes.

In uremic patients, high-density lipoprotein (HDL) loses its anti-inflammatory features and can even become pro-inflammatory due to an altered protein composition. In chronic kidney disease (CKD), impaired functions of polymorphonuclear leukocytes (PMNLs) contribute to inflammation and an increased risk of cardiovascular disease. This study investigated the effect of HDL from CKD and hemodialysis (HD) patients on the CD14 expression on PMNLs. HDL was isolated using a one-step density gradient centrifugation. Isolation of PMNLs was carried out by discontinuous Ficoll-Hypaque density gradient centrifugation. CD14 surface expression was quantified by flow cytometry. The activity of the small GTPase Rac1 was determined by means of an activation pull-down assay. HDL increased the CD14 surface expression on PMNLs. This effect was more pronounced for HDL isolated from uremic patients. The acute phase protein serum amyloid A (SAA) caused higher CD14 expression, while SAA as part of an HDL particle did not. Lipid raft disruption with methyl-ß-cyclodextrin led to a reduced CD14 expression in the absence and presence of HDL. HDL from healthy subjects but not from HD patients decreased the activity of Rac1. Considering the known anti-inflammatory effects of HDL, the finding that even HDL from healthy subjects increased the CD14 expression was unexpected. The pathophysiological relevance of this result needs further investigation.

High-Density Lipoproteins as Homeostatic Nanoparticles of Blood Plasma.

It is well known that blood lipoproteins (LPs) are multimolecular complexes of lipids and proteins that play a crucial role in lipid transport. High-density lipoproteins (HDL) are a class of blood plasma LPs that mediate reverse cholesterol transport (RCT)-cholesterol transport from the peripheral tissues to the liver. Due to this ability to promote cholesterol uptake from cell membranes, HDL possess antiatherogenic properties. This function was first observed at the end of the 1970s to the beginning of the 1980s, resulting in high interest in this class of LPs. It was shown that HDL are the prevalent class of LPs in several types of living organisms (from fishes to monkeys) with high resistance to atherosclerosis and cardiovascular disorders. Lately, understanding of the mechanisms of the antiatherogenic properties of HDL has significantly expanded. Besides the contribution to RCT, HDL have been shown to modulate inflammatory processes, blood clotting, and vasomotor responses. These particles also possess antioxidant properties and contribute to immune reactions and intercellular signaling. Herein, we review data on the structure and mechanisms of the pleiotropic biological functions of HDL from the point of view of their evolutionary role and complex dynamic nature.

High-density lipoproteins are a potential therapeutic target for age-related macular degeneration.

Strong evidence suggests that dysregulated lipid metabolism involving dysfunction of the retinal pigmented epithelium (RPE) underlies the pathogenesis of age-related macular degeneration (AMD), the leading cause of irreversible blindness in the elderly. A hallmark of AMD is the overproduction of lipid- and protein-rich extracellular deposits that accumulate in the extracellular matrix (Bruch's membrane (BrM)) adjacent to the RPE. We analyzed apolipoprotein A-1 (ApoA-1)-containing lipoproteins isolated from BrM of elderly human donor eyes and found a unique proteome, distinct from high-density lipoprotein (HDL) isolated from donor plasma of the same individuals. The most striking difference is higher concentrations of ApoB and ApoE, which bind to glycosaminoglycans. We hypothesize that this interaction promotes lipoprotein deposition onto BrM glycosaminoglycans, initiating downstream effects that contribute to RPE dysfunction/death. We tested this hypothesis using two potential therapeutic strategies to alter the lipoprotein/protein profile of these extracellular deposits. First, we used short heparan sulfate oligosaccharides to remove lipoproteins already deposited in both the extracellular matrix of RPE cells and aged donor BrM tissue. Second, an ApoA-1 mimetic, 5A peptide, was demonstrated to modulate the composition and concentration of apolipoproteins secreted from primary porcine RPE cells. Significantly, in a mouse model of AMD, this 5A peptide altered the proteomic profile of circulating HDL and ameliorated some of the potentially harmful changes to the protein composition resulting from the high-fat, high-cholesterol diet in this model. Together, these results suggest that targeting HDL interactions with BrM represents a new strategy to slow AMD progression in humans.

All You Ever Wanted to Know About APOL1 and TLFs and Did Not Dare Ask.

Interest in trypanosome lytic factors (TLFs) and apolipoprotein L1, the ion channel-forming protein component of TLFs, has increased tenfold since 2010. This is due to the association of African variants of APOL1 with kidney disease such that interest has reached circles beyond parasitology. We have extensive experience purifying and working with these proteins and protein complexes. Herein we describe our detailed purification protocols to aid the new burgeoning field by providing an opportunity for consistency in reagents used across laboratories. We emphasize that it is imperative to maintain APOL1 protein intact (~42 kDa) to analyze the active ion channel-forming component/protein.

Enrichment of HDL proteome and phospholipidome from human serum via IMAC/MOAC affinity.

High-density lipoproteins (HDLs) have anti-inflammatory and antioxidant properties and are potentially cardio-protective. Defective HDL function is caused by alterations in both the proteome and lipidome of HDL particles. As potential biomarkers, the development of analytical methods is necessary for the enrichment of HDLs. Therefore, a method for selective enrichment of HDLs using immobilized metal ion affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC) is presented. SPE-based isolation of HDLs from whole serum is adopted as an alternative to traditional ultracentrifugation methods followed by SDS-PAGE. The enrichment mechanism relies on isoelectric points of lipoproteins and metal oxide. Negatively charged lipoprotein particles interact with positively charged metal oxides and IMAC affinity, which acts as a cation. Identified proteins from HDL through MALDI-MS analysis are apo AI, AII, AIV, CI, CIII, E, J, M, H, serum amyloid A and other nonapoproteins that are part of HDL particles and perform cellular functions. This serum-based proteomics approach gives insight into the functional role of HDL. HDL-associated phospholipids have also been analyzed by LDI-MS. Results suggest that the adopted analytical strategy is a feasible idea to extract lipoproteins from serum. A comparative study of healthy and diseased samples using this approach will provide valuable information in future.

Relationship between gut microbiota and circulating metabolites in population-based cohorts.

Gut microbiota has been implicated in major diseases affecting the human population and has also been linked to triglycerides and high-density lipoprotein levels in the circulation. Recent development in metabolomics allows classifying the lipoprotein particles into more details. Here, we examine the impact of gut microbiota on circulating metabolites measured by Nuclear Magnetic Resonance technology in 2309 individuals from the Rotterdam Study and the LifeLines-DEEP cohort. We assess the relationship between gut microbiota and metabolites by linear regression analysis while adjusting for age, sex, body-mass index, technical covariates, medication use, and multiple testing. We report an association of 32 microbial families and genera with very-low-density and high-density subfractions, serum lipid measures, glycolysis-related metabolites, ketone bodies, amino acids, and acute-phase reaction markers. These observations provide insights into the role of microbiota in host metabolism and support the potential of gut microbiota as a target for therapeutic and preventive interventions.

Deepening our understanding of HDL proteome.

Introduction: High-density lipoprotein (HDL) particles are heterogeneous and their proteome is complex and distinct from HDL cholesterol. However, it is largely unknown whether HDL proteins are associated with cardiovascular protection. Areas covered: HDL isolation techniques and proteomic analyses are reviewed. A list of HDL proteins reported in 37 different studies was compiled and the effects of different isolation techniques on proteins attributed to HDL are discussed. Mass spectrometric techniques used for HDL analysis and the need for precise and robust methods for quantification of HDL proteins are discussed. Expert opinion: Proteins associated with HDL have the potential to be used as biomarkers and/or help to understand HDL functionality. To achieve this, large cohorts must be studied using precise quantification methods. Key factors in HDL proteome quantification are the isolation methodology and the mass spectrometry technique employed. Isolation methodology affects what proteins are identified in HDL and the specificity of association with HDL particles needs to be addressed. Shotgun proteomics yields imprecise quantification, but the majority of HDL studies relied on this approach. Few recent studies used targeted tandem mass spectrometry to quantify HDL proteins, and it is imperative that future studies focus on the application of these precise techniques.

Asymmetrical flow field-flow fractionation for improved characterization of human plasma lipoproteins.

High- and low-density lipoproteins (HDL and LDL) are attractive targets for biomarker discovery. However, ultracentrifugation (UC), the current methodology of choice for isolating HDL and LDL, is tedious, requires large sample volume, results in sample loss, and does not readily provide information on particle size. In this work, human plasma HDL and LDL are separated and collected using semi-preparative asymmetrical flow field-flow fractionation (SP-AF4) and UC. The SP-AF4 and UC separation conditions, sample throughput, and liquid chromatography/mass spectrometry (LC/MS) lipidomic results are compared. Over 600 µg of total proteins is recovered in a single SP-AF4 run, and Western blot results confirm apoA1 pure and apoB100 pure fractions, consistent with HDL and LDL, respectively. The SP-AF4 separation requires ~ 60 min per sample, thus providing a marked improvement over UC which can span hours to days. Lipidome analysis of SP-AF4-prepared HDL and LDL fractions is compared to UC-prepared HDL and LDL samples. Over 270 lipids in positive MS mode and over 140 lipids in negative MS mode are identified by both sample preparation techniques with over 98% overlap between the lipidome. Additionally, lipoprotein size distributions are determined using analytical scale AF4 coupled with multiangle light scattering (MALS) and dynamic light scattering (DLS) detectors. These developments position SP-AF4 as a sample preparation method of choice for lipoprotein biomarker characterization and identification. Graphical abstract ᅟ.

Dihydro-sphingosine 1-phosphate interacts with carrier proteins in a manner distinct from that of sphingosine 1-phosphate.

Dihydro-sphingosine 1-phosphate (DH-S1P) is an analog of sphingosine 1-phosphate (S1P), which is a potent lysophospholipid mediator. DH-S1P has been proposed to exert physiological properties similar to S1P. Although S1P is known to be carried on HDL via apolipoprotein M (apoM), the association between DH-S1P and HDL/apoM has not been fully elucidated. Therefore, in the present study, we aimed to elucidate this association and to compare it with that of S1P and HDL/apoM. First, we investigated the distributions of S1P and DH-S1P among lipoproteins and lipoprotein-depleted fractions in human serum and plasma samples and observed that both S1P and DH-S1P were detected on HDL; furthermore, elevated amounts of DH-S1P in serum samples were distributed to the lipoprotein-depleted fraction to a greater degree than to the HDL fraction. Concordantly, a preference for HDL over albumin was only observed for S1P, and not for DH-S1P, when the molecules were secreted from platelets. Regarding the association with HDL, although both S1P and DH-S1P prefer to bind to HDL, HDL preferentially accepts S1P over DH-S1P. For the association with apoM, S1P was not detected on HDL obtained from apoM knockout mice, while DH-S1P was detected. Moreover, apoM retarded the degradation of S1P, but not of DH-S1P. These results suggest that S1P binds to HDL via apoM, while DH-S1P binds to HDL in a non-specific manner. Thus, DH-S1P is not a mere analog of S1P and might possess unique clinical significance.

Rapid Affinity Enrichment of Human Apolipoprotein A-I Associated Lipoproteins for Proteome Analysis.

Isolation of high density lipoproteins (HDL) for structural and functional studies typically relies on ultracentrifugation techniques, which are time-consuming and difficult to scale. With emerging interest in the clinical relevance of HDL structure and function to cardiovascular disease, a significant gap exists between current and desirable sample preparation throughput. To enable proteomic studies of HDL with large clinical cohorts, we have developed an affinity enrichment approach that relies on the association of histidine-tagged, lipid free ApoA-I with HDL followed by standard metal chelate chromatography. Characterization of the resulting affinity-enriched ApoA-I associated lipoprotein (AALP) pool using biochemical, electrophoretic, and proteomic analysis demonstrates that the isolated material is closely related in structural features, lipid content, protein complement, and relative protein distribution to HDL isolated by ultracentrifugation using sequential density adjustment. The simplicity of the method provides avenues for high-throughput analysis of HDL associated proteins.

Eicosapentaenoic acid inhibits oxidation of high density lipoprotein particles in a manner distinct from docosahexaenoic acid.

The omega-3 fatty acid eicosapentaenoic acid (EPA) reduces oxidation of ApoB-containing particles in vitro and in patients with hypertriglyceridemia. EPA may produce these effects through a potent antioxidant mechanism, which may facilitate LDL clearance and slow plaque progression. We hypothesize that EPA antioxidant effects may extend to ApoA-containing particles like HDL, potentially preserving certain atheroprotective functions. HDL was isolated from human plasma and incubated at 37 °C in the absence (vehicle) or presence of EPA and/or DHA; 5.0 or 10.0 µM each. Samples were then subjected to copper-induced oxidation (10 µM). HDL oxidation was inhibited similarly by EPA and DHA up to 1 h. EPA (10 µM) maintained significant HDL oxidation inhibition of 89% (0.622 ±â€¯0.066 µM MDA; p < .001) at 4 h, with continued inhibition of 64% at 14 h, vs. vehicle (5.65 ±â€¯0.06 to 2.01 ±â€¯0.10 µM MDA; p < .001). Conversely, DHA (10 µM) antioxidant benefit was lost by 4 h. At a lower concentration (5 µM), EPA antioxidant activity remained at 81% (5.53 ±â€¯0.15 to 1.03 ±â€¯0.10 µM MDA; p < .001) at 6 h, while DHA lost all antioxidant activity by 4 h. The antioxidant activity of EPA was preserved when combined with an equimolar concentration of DHA (5 µM each). EPA pretreatment prevented HDL oxidation in a dose-dependent manner that was preserved over time. These results suggest unique lipophilic and electron stabilization properties for EPA as compared to DHA with respect to inhibition of HDL oxidation. These antioxidant effects of EPA may enhance certain atheroprotective functions for HDL.

Targeted Measurements of O- and N-Glycopeptides Show That Proteins in High Density Lipoprotein Particles Are Enriched with Specific Glycosylation Compared to Plasma.

High density lipoprotein (HDL) particles are believed to be protective due to their inverse correlation with the prevalence of cardiovascular diseases. However, recent studies show that in some conditions such as heart disease and diabetes, HDL particles can become dysfunctional. Great attention has been directed toward HDL particle composition because the relative abundances of HDL constituents determine HDL's functional properties. A key factor to consider when studying the structure and composition of plasma particles is the protein glycosylation. Here, we profile the O- and N-linked glycosylation of HDL associated-proteins including the truncated form of Apo CIII and their glycan heterogeneity in a site-specific manner. Apolipoprotein CIII, fetuin A, and alpha 1 antitrypsin are glycoproteins associated with lipoproteins and are implicated in many cardiovascular and other disease conditions. A targeted method (UHPLC-QQQ) was used to measure the glycoprotein concentrations and site-specific glycovariations of the proteins in human plasma and compared with HDL particles isolated from the same plasma samples. The proteins found in the plasma are differentially glycosylated compared to those isolated in HDL. The results of this study suggest that glycosylation may play a role in protein partitioning in the blood, with possible functional implications.

Serum amyloid A enrichment impairs the anti-inflammatory ability of HDL from diabetic nephropathy patients.

AIMS: Impaired anti-inflammatory ability of high-density lipoprotein (HDL) has been demonstrated in patients with type-2 diabetes mellitus (T2DM). However, whether HDL from patients with diabetic nephropathy (DN) suffers additional damage remains unknown. This study compared the anti-inflammatory capacities of HDL from healthy controls, T2DM patients with normal renal function, and T2DM patients with DN. MATERIALS AND METHODS: HDL was isolated from healthy controls (n=33) and T2DM patients with normal renal function (n=21), chronic kidney disease (CKD) (n=27), and end-stage renal disease (ESRD) (n=27). Human peripheral blood mononuclear cells (PBMCs) from healthy volunteers were pretreated with HDL (100µg/mL) for 1h, then incubated with lipopolysaccharide (LPS) (50ng/mL) for 24h. The anti-inflammatory ability of HDL was measured as the secretion of TNF-α in LPS-activated monocytes. RESULTS: The anti-inflammatory ability of HDL was gradually impaired as kidney function declined. Serum amyloid A (SAA) concentration in HDLDN significantly increased and was positively correlated with the impaired anti-inflammatory ability of HDL (Pearson r=0.315, P=0.006). Furthermore, HDL supplemented with SAA significantly increased TNF-α release from PBMCs compared with that from control HDL. CONCLUSIONS: These findings identified an impaired anti-inflammatory capacity of HDL from DN patients, which might be attributable to SAA enrichment.

HDL Glycoprotein Composition and Site-Specific Glycosylation Differentiates Between Clinical Groups and Affects IL-6 Secretion in Lipopolysaccharide-Stimulated Monocytes.

The goal of this pilot study was to determine whether HDL glycoprotein composition affects HDL's immunomodulatory function. HDL were purified from healthy controls (n = 13), subjects with metabolic syndrome (MetS) (n = 13), and diabetic hemodialysis (HD) patients (n = 24). Concentrations of HDL-bound serum amyloid A (SAA), lipopolysaccharide binding protein (LBP), apolipoprotein A-I (ApoA-I), apolipoprotein C-III (ApoC-III), α-1-antitrypsin (A1AT), and α-2-HS-glycoprotein (A2HSG); and the site-specific glycovariations of ApoC-III, A1AT, and A2HSG were measured. Secretion of interleukin 6 (IL-6) in lipopolysaccharide-stimulated monocytes was used as a prototypical assay of HDL's immunomodulatory capacity. HDL from HD patients were enriched in SAA, LBP, ApoC-III, di-sialylated ApoC-III (ApoC-III2) and desialylated A2HSG. HDL that increased IL-6 secretion were enriched in ApoC-III, di-sialylated glycans at multiple A1AT glycosylation sites and desialylated A2HSG, and depleted in mono-sialylated ApoC-III (ApoC-III1). Subgroup analysis on HD patients who experienced an infectious hospitalization event within 60 days (HD+) (n = 12), vs. those with no event (HD-) (n = 12) showed that HDL from HD+ patients were enriched in SAA but had lower levels of sialylation across glycoproteins. Our results demonstrate that HDL glycoprotein composition, including the site-specific glycosylation, differentiate between clinical groups, correlate with HDL's immunomodulatory capacity, and may be predictive of HDL's ability to protect from infection.

A novel approach to measuring macrophage-specific reverse cholesterol transport in vivo in humans.

Reverse cholesterol transport (RCT) is thought to be an atheroprotective function of HDL, and macrophage-specific RCT in mice is inversely associated with atherosclerosis. We developed a novel method using 3H-cholesterol nanoparticles to selectively trace macrophage-specific RCT in vivo in humans. Use of 3H-cholesterol nanoparticles was initially tested in mice to assess the distribution of tracer and response to interventions known to increase RCT. Thirty healthy subjects received 3H-cholesterol nanoparticles intravenously, followed by blood and stool sample collection. Tracer counts were assessed in plasma, nonHDL, HDL, and fecal fractions. Data were analyzed by using multicompartmental modeling. Administration of 3H-cholesterol nanoparticles preferentially labeled macrophages of the reticuloendothelial system in mice, and counts were increased in mice treated with a liver X receptor agonist or reconstituted HDL, as compared with controls. In humans, tracer disappeared from plasma rapidly after injection of nanoparticles, followed by reappearance in HDL and nonHDL fractions. Counts present as free cholesterol increased rapidly and linearly in the first 240 min after nadir; counts in cholesteryl ester increased steadily over time. Estimates of fractional transfer rates of key RCT steps were obtained. These results support the use of 3H-cholesterol nanoparticles as a feasible approach for the measurement of macrophage RCT in vivo in humans.

Stability of serum high-density lipoprotein-microRNAs for preanalytical conditions.

Background Recently, several studies have shown that microRNAs are present in high-density lipoprotein, and high-density lipoprotein-microRNA may be a promising disease biomarker. We investigated the stability of high-density lipoprotein-microRNAs in different storage conditions as this is an important issue for its application to the field of clinical research. Methods microRNAs were extracted from the high-density lipoprotein fraction that was purified from the serum. miR-135 a and miR-223, which are known to be present in high-density lipoprotein, were quantified by quantitative real-time PCR. The influence of preanalytical parameters on the analysis of high-density lipoprotein-miRNAs was examined by the effect of RNase, storage conditions, and freezing and thawing. Results The concentrations of microRNA in high-density lipoprotein were not altered by RNase A treatment (0-100 U/mL). No significant change in these microRNAs was observed after storing serum at room temperature or 4℃ for 0-24 h, and there was a similar result in the cryopreservation for up to two weeks. Also, high-density lipoprotein-microRNAs were stable for, at least, up to five freeze-thaw cycles. Conclusions These results demonstrated that high-density lipoprotein-microRNAs are relatively resistant to various storage conditions. This study provides new and important information on the stability of high-density lipoprotein-microRNAs.

Infrared analysis of lipoproteins in the detection of alcohol biomarkers.

BACKGROUND: Alcoholism is a major public health problem. Alcohol causes modifications in the composition and concentration of lipoproteins and influences the enzymes and transfer proteins that transform lipoproteins in plasma. Alcohol is associated with the presence of alcohol biomarkers (fatty acid ethyl esters [FAEEs] and phosphatidylethanol [PEth]) in lipoproteins. We explore the possibilities of detecting alcohol biomarkers in non-high-density-lipoproteins (non-HDLs) precipitated from serum using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). METHODS: Analyzes were carried out on stored serum samples, with known % carbohydrate-deficient transferrin (CDT) values, included in a driver's license regranting program under the control of the Belgian Institute of Road Safety. The study consisted of 127 control samples (CDT≤1.3%) and 114 alcoholic samples (CDT>1.3%). Liver enzymes, CRP, triglycerides, total, HDL- and LDL-cholesterol values were determined. Non-HDLs were precipitated with sodium phosphotungstate and MgCl2 and analyzed using ATR-FTIR in the range from 4500 cm-1 to 450 cm-1 using a Perkin Elmer ATR-FTIR Spectrometer Two. RESULTS: The area under the curve of the 1130-990 cm-1 region (AUC1130-990 cm-1) was able to discriminate controls from alcoholics (p<0.0001) due to the presence of FAEEs in lipoproteins. Multiple regression analysis significantly predicted the AUC1130-990 cm-1 (adj. r2=0.13, p<0.0001). Significant correlations were found between AUC1130-990 cm-1 and CDT values (r=0.32, p<0.0001), AST/ALT ratio (r=0.21, p=0.001). GGT showed no significant correlation. CONCLUSIONS: Infrared analysis of lipoproteins is a potential tool in the detection of alcohol biomarkers.

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification.

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most interestingly, sRNAs are also found outside of cells and are stably present in all biological fluids. As such, extracellular sRNAs represent a novel class of disease biomarkers and are likely involved in cell signaling and intercellular communication networks. To assess their potential as biomarkers, sRNAs can be quantified in plasma, urine, and other fluids. Nevertheless, to fully understand the impact of extracellular sRNAs as endocrine signals, it is important to determine which carriers are transporting and protecting them in biological fluids (e.g., plasma), which cells and tissues contribute to extracellular sRNA pools, and cells and tissues capable of accepting and utilizing extracellular sRNA. To accomplish these goals, it is critical to isolate highly pure populations of extracellular carriers for sRNA profiling and quantification. We have previously demonstrated that lipoproteins, particularly high-density lipoproteins (HDL), transport functional microRNAs (miRNA) between cells and HDL-miRNAs are significantly altered in disease. Here, we detail a new protocol that utilizes tandem HDL isolation with density-gradient ultracentrifugation (DGUC) and fast-protein-liquid chromatography (FPLC) to obtain highly pure HDL for downstream profiling and quantification of all sRNAs, including miRNAs, using both high-throughput sequencing and real-time PCR approaches. This protocol will be a valuable resource for the investigation of sRNAs on HDL.

Refined purification strategy for reliable proteomic profiling of HDL2/3: Impact on proteomic complexity.

Proteomics have extended the list of high-density lipoprotein (HDL) associated proteins to about 90. One of the major issues of global protein characterization is establishing specificity of association as opposed to contamination, a fact which has never been addressed for isolated HDL. We have developed a refined purification strategy to isolate HDL by density, followed by purification by size to generate "highly purified" fractions of HDL2/3, which allow the reliable quantification of the HDL proteome for biomarker discovery. Mass spectrometry analysis revealed that the proteome of HDL2/3 is composed of 10-16 different proteins, which is in striking contrast to previous reports. Importantly, proteomic analysis revealed that many proteins which have recently been described to be associated with HDL, including α-1-antitrypsin, α-2-HS-glycoprotein, serotransferrin, apolipoprotein A-IV and others, are not associated with HDL2/3 and are exclusively found in a different molecular weight fraction containing human serum albumin, lipid-poor apolipoprotein A-I and other proteins. Interestingly, proteins found in this lower molecular weight fraction commonly share lipid-binding properties and enrichment of serum with free fatty acids/lysophophatidylcholine led to a significant increase in co-isolation of lipid-binding proteins such as albumin and α-1-antitrypsin. We propose that this refined method might become a standard in proteomic assessment of HDL2/3 making data from clinical cohorts more comparable and reproducible.