Timothy Collier (Presenter)
Cleveland Heartlab, Inc.
Bio: Currently a Senior Research and Development Scientist at Cleveland Heartlab, Inc. Obtained Ph.D. from NC State University in 2010 (Dave Muddiman's Research Group) Post Doc 2010-2013 at Washington University (in St. Louis) School of Medicine in Molecular Oncology
Authorship: Timothy S. Collier, Zhicheng Jin, Celalettin Topbas, and Cory Bystrom
Cleveland Heartlab, Inc.
The purification of high density lipoprotein (HDL) for structural and functional studies typically utilizes ultracentrifugation that is time-consuming and challenging to scale-up for large clinical studies. As interest increases in the relevance of HDL composition and function as it relates to cardiovascular health, a significant need exists for high-throughput methodologies. We present an approach which relies on association of recombinant His-tagged ApoA-I with HDL, allowing subsequent immobilized metal affinity purification of ApoA-I associated high density lipoproteins (AA-HDL). The enrichment procedure takes minutes and provides a pool AA-HDL which retains close agreement in structure, composition, and enzymatic activity to HDL isolated by ultracentrifugation. Additionally, the use of mild elution conditions yields a sample readily amenable for proteo-/metabolomic MS analysis.
High density lipoprotein (HDL) is a complex, biologically active macromolecular complex whose range of function includes lipid and cholesterol transport, anti-oxidative and anti-thrombotic activity, endothelial regulation, and immune response regulation. The anti-atherogenic properties of HDL are ascribed to several of these functions, which are increasingly explored using sensitive mass spectrometry based techniques. Mass spectrometry analysis of HDL requires techniques that can isolate it from serum, typically density gradient ultracentrifugation. This process is time-consuming, difficult to scale, and requires extensive post-isolation treatment to yield a sample that is amenable to mass spectrometric characterization. Here, we present a rapid, scalable method for isolation of ApoA-I associated high density lipoproteins (AA-HDL) under gentle buffer conditions. This method uses recombinant, histidine-tagged Apolipoprotein A-I (ApoA-I) and takes advantage of the affinity of lipid-free ApoA-I for HDL allowing for subsequent particle isolation by immobilized metal affinity chromatography (IMAC). Characterization of AA-HDL demonstrates a very close structural, compositional, and quantitative relationship to HDL isolated by ultracentrifugation.
Recombinantly expressed and purified His-tagged ApoA-I was combined with serum in a range of molar ratios (His6ApoA-I to Endogenous ApoA-I) ranging from 1:10 to 10:1 and incubated at 37oC up to 2 hours to determine optimum isolation conditions. Purification is achieved on metal chelating paramagnetic beads or pipet tips containing an IMAC column bed according to the manufacturers protocol. For proteomic analysis, eluted lipoproteins were treated with 5 mM DTT and digested with the addition of Lys-C and incubation at 37oC for up to 4 hours.
The HDL proteome was surveyed by injecting ~500 ng of total peptide material and analyzing by LC-MS/MS using a Waters nanoACQUITY LC directly coupled to an Thermo Orbitrap Velos mass spectrometer operating at 120,000 resolving power. Proteins were identified using the Andromeda search engine in MaxQuant (v220.127.116.11) against the Uniprot Human Database. Targeted MRM analysis of known HDL-associated proteins was accomplished by injecting 10 µL of LysC digested material using an Agilent 1260 HPLC system and detecting with an Agilent 6490 triple quadrupole mass spectrometer operating in dynamic MRM mode. Transitions determined to be unique to the peptide targeted in the sample were selected and optimized using Skyline. Two transitions were monitored per peptide, and up to two peptides monitored per protein. Peptides were quantified against a well characterized, stable isotope-labelled internal standard peptide mixture using MassHunter Quantitative analysis software.
HDL particle analysis was accomplished using microfluidic separation on the Agilent 2100 Bioanalyzer according to a custom protocol for HDL subfractionation. Briefly, HDL samples are diluted and incubated in the presence of lipophilic dye and loaded into appropriate wells on a microfluidic chip. During separation, detection is by laser induced fluorescence at 680 nm.
Phospholipid, triglyceride, total cholesterol and free cholesterol content were measured using commercially available enzymatic assays (CVs <10%). HDL paraoxonase activity was assessed using the EnzChek Paraoxonase Assay Kit according to the manufacturer’s protocol.
Our experiments showed that AA-HDL is efficiently isolated from human serum after incubation with recombinant His6ApoA-I and subsequent purification using immobilized metal affinity chromatography, with endogenous ApoA-I recovery of approximately 50% from a ~10 µL sample, with optimum yield occurring near a 1:1 molar ratio of exogenous to endogenous ApoA-I. Anti-His tag western blots of non-denaturing SDS-PAGE gels show efficient incorporation of recombinant ApoA-I into HDL-sized species. Further investigation of HDL subclasses analysis suggests that the addition of supra-physiological levels of His6ApoA-I to serum did not result in significant shifts in the relative proportions of HDL2 and HDL3 populations. The relative amounts of lipid classes, including phospholipids, triglycerides, total and free cholesterol in enriched AA-HDL particles was consistent with those of HDL prepared by ultracentrifugation. Additionally, the weight percentages of each lipid class were consistent with values reported in the literature for HDL. A comparison of the proteomes between HDL samples prepared by either ApoA-I affinity or ultracentrifugation yielded very similar proteomes. Of the 95 proteins identified in 3 or more previous HDL proteome surveys in the literature, 88 were identified in affinity-isolated HDL, with 80 identified in ultracentrifuged HDL, and 77 proteins shared between both techniques. Targeted MRM analysis of several HDL-associated proteins revealed strong correlations between enrichment strategies, with Pearson r values ranging from 0.81 to 0.99. Furthermore, the AALPs retained paraoxonase activity that strongly correlates with the activity of their originating neat serum samples (Spearman r = 0.73).
Conclusions & Discussion
The evaluation of enriched AA-HDL indicates a very close structural and compositional relationship to HDL isolated by ultracentrifugation. Affinity preparation of AA-HDL is achievable in minutes, in contrast to hours to days necessary for UC. Furthermore, the sample is obtained in a matrix suitable for immediate use in mass spectrometry workflows, without the need for extensive dialysis to remove density-gradient reagents.
References & Acknowledgements:
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IP Royalty: no
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