L. Jason Lu

Proteomic Characterization of Human Plasma High Density Lipoprotein Fractionated by Gel Filtration Chromatography

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.

The Effects of Type 2 Diabetes on Lipoprotein Composition and Arterial Stiffness in Male Youth

Recent studies suggest HDL exists as numerous subpopulations with distinct protein/lipid compositions that are not reflected in the HDL cholesterol (HDL-C) number. In this study, we sought to evaluate HDL subpopulations in adolescents with type 2 diabetes (T2D) to determine if changes in HDL composition are associated with early vascular disease. T2D (n = 10), lean (n = 9), and obese (n = 11) youth were recruited. Plasma was fractionated using gel-filtration chromatography, and lipid-associated proteins were identified using mass spectrometry. Concurrently, vascular stiffness was assessed using pulse wave velocity (PWV). We found youth with T2D exhibited decreased phospholipid content in fractions containing large HDL particles that was inversely associated with PWV (P < 0.001). No association was noted between HDL-C and PWV. Proteomic analysis revealed changes in 7 of 45 identified proteins in the T2D group, including apolipoprotein (apo) A-II, apoE, and paraoxonase-1 (P < 0.05). Our data demonstrate early changes in the lipid and protein compositions of specific HDL subspecies in adolescents with T2D that are related to early markers of arterial disease. These findings suggest that analyzing the composition of HDL, rather than HDL-C, may be useful in assessing cardiovascular risk in this population.

Multi-dimensional Co-separation Analysis Reveals Protein–Protein Interactions Defining Plasma Lipoprotein Subspecies

The distribution of circulating lipoprotein particles affects the risk for cardiovascular disease (CVD) in humans. Lipoproteins are historically defined by their density, with low-density lipoproteins positively and high-density lipoproteins (HDLs) negatively associated with CVD risk in large populations. However, these broad definitions tend to obscure the remarkable heterogeneity within each class. Evidence indicates that each class is composed of physically (size, density, charge) and compositionally (protein and lipid) distinct subclasses exhibiting unique functionalities and differing effects on disease. HDLs in particular contain upward of 85 proteins of widely varying function that are differentially distributed across a broad range of particle diameters. We hypothesized that the plasma lipoproteins, particularly HDL, represent a continuum of phospholipid platforms that facilitate specific protein–protein interactions. To test this idea, we separated normal human plasma using three techniques that exploit different lipoprotein physicochemical properties (gel filtration chromatography, ionic exchange chromatography, and preparative isoelectric focusing). We then tracked the co-separation of 76 lipid-associated proteins via mass spectrometry and applied a summed correlation analysis to identify protein pairs that may co-reside on individual lipoproteins. The analysis produced 2701 pairing scores, with the top hits representing previously known protein–protein interactions as well as numerous unknown pairings. A network analysis revealed clusters of proteins with related functions, particularly lipid transport and complement regulation. The specific co-separation of protein pairs or clusters suggests the existence of stable lipoprotein subspecies that may carry out distinct functions. Further characterization of the composition and function of these subspecies may point to better targeted therapeutics aimed at CVD or other diseases.

A Comparison of the Mouse and Human Lipoproteome: Suitability of the Mouse Model for Studies of Human Lipoproteins

Plasma levels of low density lipoproteins (LDL) and high density lipoproteins (HDL) exhibit opposing associations with cardiovascular disease in human populations and mouse models have been heavily used to derive a mechanistic understanding of these relationships. In humans, recent mass spectrometry studies have revealed that the plasma lipoproteome is significantly more complex than originally appreciated. This is particularly true for HDL which contains some 90 distinct proteins, a majority of which play functional roles that go beyond those expected for simple lipid transport. Unfortunately, the mouse lipoproteome remains largely uncharacterized-a significant gap given the heavy reliance on the model. Using a gel filtration chromatography and mass spectrometry analysis that targets phospholipid-bound plasma proteins, we compared the mouse lipoproteome and its size distribution to a previous, identical human analysis. We identified 113 lipid associated proteins in the mouse. In general, the protein diversity in the LDL and HDL size ranges was similar in mice versus humans, though some distinct differences were noted. For the majority of proteins, the size distributions, that is, whether a given protein was associated with large versus small HDL particles, for example, were also similar between species. Again, however, there were clear differences exhibited by a minority of proteins that may reflect metabolic differences between species. Finally, by correlating the lipid and protein size profiles, we identified five proteins that closely track with the major HDL protein, apolipoprotein A-I across both species. Thus, mice have most of the minor proteins identified in human lipoproteins that play key roles in inflammation, innate immunity, proteolysis and its inhibition, and vitamin transport. This provides support for the continued use of the mouse as a model for many aspects of human lipoprotein metabolism.

Network-Based Analysis on Orthogonal Separation of Human Plasma Uncovers Distinct High Density Lipoprotein Complexes

High density lipoprotein (HDL) particles are blood-borne complexes whose plasma levels have been associated with protection from cardiovascular disease (CVD). Recent studies have demonstrated the existence of distinct HDL subspecies; however, these have been difficult to isolate and characterize biochemically. Here, we present the first report that employs a network-based approach to systematically infer HDL subspecies. Healthy human plasma was separated into 58 fractions using our previously published three orthogonal chromatography techniques. Similar local migration patterns among HDL proteins were captured with a novel similarity score, and individual comigration networks were constructed for each fraction. By employing a graph mining algorithm, we identified 183 overlapped cliques, among which 38 were further selected as candidate HDL subparticles. Each of these 38 subparticles had at least two literature supports. In addition, GO function enrichment analysis showed that they were enriched with fundamental biological and CVD protective functions. Furthermore, gene knockout experiments in mouse model supported the validity of these subparticles related to three apolipoproteins. Finally, analysis of an apoA-I deficient human patients plasma provided additional support for apoA-I related complexes. Further biochemical characterization of these putative subspecies may facilitate the mechanistic research of CVD and guide targeted therapeutics aimed at its mitigation.

Mapping Atheroprotective Functions and Related Proteins/Lipoproteins in Size Fractionated Human Plasma

HDL has been shown to possess a variety of cardio-protective functions, including removal of excess cholesterol from the periphery, and inhibition of lipoprotein oxidation. It has been proposed that various HDL subparticles exist, each with distinct protein and lipid compositions, which may be responsible for HDLs many functions. We hypothesized that HDL functions will co-migrate with the operational lipoprotein subspecies when separated by gel filtration chromatography. Plasma from 10 healthy male donors was fractionated and the protein composition of the phospholipid containing fractions was analyzed by mass spectrometry (MS). Each fraction was evaluated for its proteomic content as well as its ability to promote cholesterol efflux and protect low density lipoprotein (LDL) from free radical oxidation. For each function, several peaks of activity were identified across the plasma size gradient. Neither cholesterol efflux or LDL antioxidation activity correlated strongly with any single protein across the fractions. However, we identified multiple proteins that had strong correlations (r values >0.7, p < 0.01) with individual peaks of activity. These proteins fell into diverse functional categories, including those traditionally associated with lipid metabolism, as well as alternative complement cascade, innate immunity and clotting cascades and immunoglobulins. Additionally, the phospholipid and cholesterol concentration of the fractions correlated strongly with cholesterol efflux (r = 0.95 and 0.82 respectively), whereas the total protein content of the fractions correlated best with antioxidant activity across all fractions (r = 0.746). Furthermore, two previously postulated subspecies (apoA-I, apoA-II and apoC-1; as well as apoA-I, apoC-I and apoJ) were found to have strong correlations with both cholesterol efflux and antioxidation activity. Up till now, very little has been known about how lipoprotein composition mediates functions like cholesterol efflux and antioxidation.

A Comparison of Methods To Enhance Protein Detection of Lipoproteins by Mass Spectrometry

We sought to develop a new method to more efficiently analyze lipid-bound proteins by mass spectrometry using a combination of a lipid removal agent (LRA) that selectively targets lipid-bound proteins and a mass spectrometry compatible detergent, anionic acid labile surfactant (AALS), that is capable of eluting proteins off the LRA. This method was compared to established methods that use the lipid removal agent alone and straight proteomic analysis of human plasma after organic solvent delipidation (OSD). Plasma from healthy individuals was separated by gel filtration chromatography and prepared for mass spectrometry analysis by each of the described methods. The addition of AALS to LRA increased the overall number of proteins detected in both the high and low density lipoprotein size range, the number of peptide counts for each protein, and the overall sequence coverage. Organic solvent delipidation detected the most proteins, though with some decrease in overall protein detection and sequence coverage due to the presence of nonlipid-bound proteins. The use of LRA allows for selection and analysis of lipid-bound proteins. The addition of a mass spectrometry compatible detergent improved detection of lipid-bound proteins from human plasma using LRA.

High-Density Lipoproteins-Associated Proteins and Subspecies Related to Arterial Stiffness in Young Adults with Type 2 Diabetes Mellitus

Lower plasma levels of high-density lipoproteins (HDL) in adolescents with type 2 diabetes (T2D) have been associated with a higher pulse wave velocity (PWV), a marker of arterial stiffness. Evidence suggests that HDL proteins or particle subspecies are altered in T2D and these may drive these relationships. In this work, we set out to reveal any specific proteins and subspecies that are related to arterial stiffness in youth with T2D from proteomics data. Plasma and PWV measurements were previously acquired from lean and T2D adolescents. Each plasma sample was separated into 18 fractions and evaluated by mass spectrometry. Then, we applied a validated network-based computational approach to reveal HDL subspecies associated with PWV. Among 68 detected phospholipid-associated proteins, we found that seven were negatively correlated with PWV, indicating that they may be atheroprotective. Conversely, nine proteins show positive correlation with PWV, suggesting that they may be related to arterial stiffness. Intriguingly, our results demonstrate that apoA-I and histidine-rich glycoprotein may reverse their protective roles and become antagonistic in the setting of T2D. Furthermore, we revealed two arterial stiffness-associated HDL subspecies, each of which contains multiple PWV-related proteins. Correlation and disease association analyses suggest that these HDL subspecies might link T2D to its cardiovascular-related complications.

Erratum. The Effects of Type 2 Diabetes on Lipoprotein Composition and Arterial Stiffness in Male Youth. Diabetes 2013;62:2958–2967

In the article listed above, there is an error in Fig. 1 B . The authors recently noted an …