1 Rapid Affinity Enrichment of Human Apolipoprotein A-I Associated

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Rapid Affinity Enrichment of Human Apolipoprotein A-I Associated Lipoproteins for Proteome Analysis Timothy S Collier, Zhicheng Jin, Celalettin Topbas, and Cory Bystrom J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00816 • Publication Date (Web): 07 Feb 2018 Downloaded from http://pubs.acs.org on February 7, 2018

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Journal of Proteome Research

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

Timothy S. Collier1, Zhicheng Jin1, Celalettin Topbas1, and Cory Bystrom1*

1. Cleveland HeartLab, Inc. 6701 Carnegie Avenue, Suite 500 Cleveland, OH 44103

CORRESPONDING AUTHOR: Cory Bystrom Cleveland HeartLab, Inc. 6701 Carnegie Avenue, Suite 500 Cleveland, OH 44103 Telephone: (216) 426-6081, extension: 1406 Fax: (866) 869-0148 [email protected]

1 ACS Paragon Plus Environment

Journal of Proteome Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABBREVIATIONS: Tris, Borate, EDTA Buffer (TBE); Polyacrylamide gel electrophoresis (PAGE); Ultracentrifugation (UC); Phosphate buffered saline (PBS);

ABSTRACT: 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 which 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.

KEYWORDS: Affinity Enrichment, Targeted Proteomics, High-Density Lipoprotein, HighThroughput, Serum


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INTRODUCTION: High Density Lipoproteins (HDL) are ensembles of biologically active macromolecular structures with a diverse range of functions.

Lipid and cholesterol transport1,2, antioxidative3,

antithrombotic, endothelial and immune regulating4 activities of HDL have been characterized and many have been associated with anti-atherogenic and anti-inflammatory properties of HDL. Detailed functional studies of HDL have accelerated with the availability of sensitive mass spectrometry based proteomic and metabolomic platforms which have expanded the depth of qualitative characterization and provided the ability to perform quantitative comparisons

5–7

. The

understanding of HDL as an active complex has grown concomitantly with studies that have cataloged the proteome and advanced toward quantitative studies of HDL and alterations in composition that are associated with cardiovascular disease and metabolic dysfunction.

Historically, HDL has been defined and prepared by density ultracentrifugation (UC) where the density of total HDL is 1.063-1.25 g/mL with two major sub-fractions, HDL2 (d=1.063 to 1.125g/mL) and HDL3 (d=1.125 to 1.21 g/mL). However, it is also recognized that HDL has been defined by apolipoprotein content, size, surface charge, or a combination of these characteristics as revealed by immunoaffinity and/or electrophoretic mobility in various matrices 8–12

. Application of proteomic and metabolomic techniques to the analysis of lipoproteins

requires techniques which can enrich the complexes from abundant serum proteins.

For

laboratory scale preparations, ultracentrifugation is an effective and well characterized technique and is widely considered “gold standard” although many different types of ultracentrifugation have been employed; multi-step density adjustment as well as gradient methods have been successfully employed all in a range of rotor geometries and centrifuge times.

Size exclusion chromatography12 and immunoaffinity preparations13 have also been

reported but have not been as widely adopted. 3 ACS Paragon Plus Environment


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As a preparative technique, UC it is not without limitations. The range of differences in the application of the technique can leave HDL preparations with distinct characteristics.

In

particular, shear stress and ionic strength dependent changes in HDL composition have been reported. The most widely used preparative method fractionation requires many hours of centrifugal separation in the presence of high salt concentrations during which time the particles are exposed to non-physiological conditions with the risk of oxidative damage, protein loss and particle remodeling14,15. The technique is difficult to scale as sample throughput requirements grow with constraints arising from centrifuge rotor capacity, time of preparation and typical requirements for post-isolation purification to remove the density gradient agent (typically potassium bromide or sucrose).

Methods such as size exclusion chromatography or

immunoaffinity approaches conceivably overcome one or more of these issues but each brings limitations of their own and are not yet broadly accepted. In the case of size exclusion chromatography, pooled fractions do not contain lipoproteins alone, necessitating further selective isolation procedures12. In the case of immunoaffinity enrichment, literature reports suggest that ApoA-I containing complexes are somewhat different than UC prepared HDL. In addition, known post-translational modifications of ApoA-I could conceivably interfere with immunoaffinity isolation of a representative pool of lipoproteins 10,16,17.

With the growing interest in studies focused on HDL structure, function, and composition as it relates to human health, there remains a need for techniques that can rapidly enrich ApoA-I containing lipoprotein particles in a manner suitable for high-throughput application of “omic” analyses. To address this challenge, we have developed a method of enriching ApoA-I associated lipoproteins (AALP) which takes advantage of the affinity of lipid free ApoA-I for HDL18,19 to incorporate an affinity tag. Here we describe the method of enrichment and provide


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a comparison of the proteome of these isolates to HDL isolated by UC using sequential density adjustment with potassium bromide.

EXPERIMENTAL SECTION: Unless otherwise specified, all reagents were purchased from Sigma-Aldrich (St. Louis, MO) or ThermoFisher (Waltham, MA) at the highest available quality. TBE buffered 4-20% PAGE gels were purchased from Invitrogen (Waltham, MA). Commercially sourced high-purity HDL in phosphate buffered saline (isolated by ultracentrifugation from potassium bromide) was sourced from Athens Research (Athens, GA). Specimens used in this study were obtained from Golden West Biologicals (Temecula, CA) which supplied pooled serum samples. Additionally, remnant samples from Cleveland HeartLab were used. immunoaffinity

chromatography

Recombinantly expressed,

was

Lipoprotein depleted serum prepared via

purchased

from

GenwayBio

(San

Diego,

CA).

15

N-labelled, His6-tagged Apolipoprotein A-I (15N-His6ApoA-I) was

purchased from Genscript (Piscataway, NJ). Purity of this material was determined to be >90% based on gel electrophoresis and LC-UV/Vis-MS.

An equivalent product is available from

Cambridge Isotopes.

Gel Separation and Western Blot UC-prepared HDL, commercially sourced or prepared in-house, was diluted to 1.5 mg/ml with PBS. 30 µL of HDL stock was mixed with 60 µL of 0.5 mg/mL lipid free recombinant

15

N-

His6ApoA-I or with PBS and incubated at 37°C for 20 minutes. Equivalent amounts of protein based on Bradford assay (sufficient for staining or blotting) were separated on a 4-20% gradient non-denaturing TBE gel (170 V constant, 3 hours, 4°C). Gel was transferred to polyvinylidene difluoride membrane and probed with HRP conjugated anti-His6 antibody (Clontech, Mountain View, CA) and HRP conjugated anti-ApoA-I antibody (Academy Bio-Medical Company, 5 ACS Paragon Plus Environment


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Houston, TX). Stripping and re-probing of the membrane (1.5 % Glycine, 0.1% SDS, 1% Tween-20, pH 2.2) was used for multiple detections. The blots were imaged by chemiluminescence (Pierce, Waltham, MA).

Isolation of HDL by Ultracentrifugation HDL (1.063 g/mL

1 Rapid Affinity Enrichment of Human Apolipoprotein A-I Associated

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