Proteomic characterization of human plasma high density lipoprotein fractionated by gel filtration chromatography

Abstract

Plasma levels of high density lipoprotein cholesterol (HDL-C) are inversely proportional to the incidence of cardiovascular disease. Recent applications of modern proteomic technologies have identified upward of 50 distinct proteins associated with HDL particles with many of these newly discovered proteins implicating HDL in nonlipid transport processes including complement activation, acute phase response and innate immunity. However, almost all MS-based proteomic studies on HDL to date have utilized density gradient ultracentrifugation techniques for HDL isolation prior to analysis. These involve high shear forces and salt concentrations that can disrupt HDL protein interactions and alter particle function. Here, we used high-resolution size exclusion chromatography to fractionate normal human plasma to 17 phospholipid-containing subfractions. Then, using a phospholipid binding resin, we identified proteins that associate with lipoproteins of various sizes by electrospray ionization mass spectrometry. We identified 14 new phospholipid-associated proteins that migrate with traditionally defined HDL, several of which further support roles for HDL in complement regulation and protease inhibition. The increased fractionation inherent to this method allowed us to visualize HDL protein distribution across particle size with unprecedented resolution. The observed heterogeneity across subfractions suggests the presence of HDL particle subpopulations each with distinct protein components that may prove to impart distinct physiological functions.

Figure 1

Elution profiles from Superose 6 (2×) and Superdex 200 (3×) size exclusion chromatography configurations. Three-hundred seventy microliters of fresh human plasma from a normal male donor was analyzed by a tandem Superose 6 setup (a) or a triple Superdex setup (b) as described in the Experimental section. Total protein (●, determined by Lowry assay) and total cholesterol (○, enzymatic assay) profiles across the fractions are shown. Peak designations refer to 1, VLDL; 2, LDL; 3, HDL; and 4, lipid-free plasma proteins.

Figure 2

SDS PAGE comparison of total HDL preparations derived from ultracentrifugation (UC) and gel filtration (GF) chromatography. A 4–15% SDS-PAGE analysis of total HDL isolated by UC (lane 1) or pooled HDL fractions from the triple Superdex 200 gel filtration separation (lane 2) is shown. The gel was stained with coomassie blue.

Figure 3

Ability of calcium silicate hydrate (CSH) to bind ultracentrifugally isolated human plasma lipoproteins. UC isolated LDL (a) or HDL (b) were analyzed by SDS-PAGE prior to incubation with CSH (lane 1 of each panel). The resulting flow through is shown in lane 2 and the proteins retained on the resin after boiling with SDS sample buffer is shown in lane 3 of each panel. The gels were stained with coomassie blue. (c) Same experiment as in (b), except that apoA-I was detected by Western blot using an antihuman apoA-I antibody.

Figure 4

Ability of CSH to bind phospholipid-containing particles from fractions collected by gel filtration chromatography. (a) Two identical samples of human plasma were fractionated on the triple Superdex gel filtration set up. One set of fractions was incubated with CSH under the conditions described in the Experimental section (●) while the other fraction was left untreated (○). The traces show the phospholipid content of each fraction as determined by enzymatic assay. (b) Triple Superdex gel filtration fraction 23 (lane 1) was incubated with CSH for 30 min and then the supernatant containing unbound components was removed (lane 2). The CSH was washed with buffer and bound proteins were recovered from CSH by boiling in SDS-sample buffer (lane 3). SDS-PAGE was carried out on a 4–15% gel and stained with coomassie brilliant blue. (c) ApoB containing lipoproteins (fraction 16) were analyzed in the same manner as described for (b).

Figure 5

Examples of elution profile shifts for proteins upon ether delipidation of fresh human plasma. Protein distribution profiles of selected proteins from untreated (○) and ether delipidated plasma (●) after separation by the triple Superdex setup. The distribution of each protein is represented as spectral count per fraction measured by ESI–MS/MS. (a) Plasminogen, which fails to exhibit a molecular size shift in response to ether delipidation and therefore is not associated with lipid, and (b) complement C3 which does shift, indicating an association with lipid. Representative data shown from two independent experiments are shown.

Figure 6

Lipid-associated proteins identified in the plasmas of 3 normolipidemic donors. The proteins included in this list met all identification criteria laid out in the Experimental section and showed a shift in elution pattern after ether delipidation, indicating lipid association. The proteins are arranged according to the sum of identified peptides across all gel filtration fractions for all 3 subjects. Proteins indicated with an asterisk have not been previously described as lipid associated proteins, to our knowledge.

Figure 7

Distribution patterns of common HDL associated proteins across gel filtration fractions. For each protein, the number of spectral counts in a given fraction is represented by bar height. The values represent the sum of counts from 3 subjects.

Figure 8

Triple Superdex distribution profiles for identified lipid-associated proteins. For each fraction, the relative abundance (determined by peptide count) is shown. A value of 1.0 was assigned to the fraction containing the highest peptide count for that particular protein and all other fractions were scaled from there. The relative abundance of each can also be assessed by the color of the square with blue representing 0 detected peptides and red representing the highest number.

Figure 9

Gene Ontology functional associations of newly identified lipoprotein associated proteins. Identified proteins are grouped by functional category (left column) and enrichment of a particular function for either newly identified or previously established proteins is presented as the number of proteins possessing function divided by total number of proteins in group. P values are given in parentheses.