Portal de Periódicos da CAPES

Proteomic diversity of high density lipoproteins: our emerging understanding of its importance in lipid transport and beyond

2013; Elsevier BV; Volume: 54; Issue: 10 Linguagem: Inglês

10.1194/jlr.r035725

1539-7262

Amy S. Shah, Lirong Tan, Jason Lu Long, W. Sean Davidson,

Cancer, Lipids, and Metabolism

Recent applications of mass spectrometry technology have dramatically increased our understanding of the proteomic diversity of high density lipoproteins (HDL). Depending on the method of HDL isolation, upwards of 85 proteins have been identified, and the list continues to grow. In addition to proteins consistent with traditionally accepted roles in lipid transport, HDL carries surprising constituents, such as members of the complement pathway, protease inhibitors involved in hemostasis, acute-phase response proteins, immune function mediators, and even metal-binding proteins. This compositional diversity fits well with hundreds of studies demonstrating a wide functional pleiotrophy, including roles in lipid transport, oxidation, inflammation, hemostasis, and immunity. This review summarizes the progression of our understanding of HDL proteomic complexity and points out key experimental observations that reinforce the functional diversity of HDL. The possibility of specific HDL subspecies with distinct functions, the evidence supporting this concept, and some of the best examples of experimentally defined HDL subspecies are also discussed. Finally, key challenges facing the field are highlighted, particularly the need to identify and define the function of HDL subspecies to better inform attempts to pharmacologically manipulate HDL for the benefit of cardiovascular disease and possibly other maladies. Recent applications of mass spectrometry technology have dramatically increased our understanding of the proteomic diversity of high density lipoproteins (HDL). Depending on the method of HDL isolation, upwards of 85 proteins have been identified, and the list continues to grow. In addition to proteins consistent with traditionally accepted roles in lipid transport, HDL carries surprising constituents, such as members of the complement pathway, protease inhibitors involved in hemostasis, acute-phase response proteins, immune function mediators, and even metal-binding proteins. This compositional diversity fits well with hundreds of studies demonstrating a wide functional pleiotrophy, including roles in lipid transport, oxidation, inflammation, hemostasis, and immunity. This review summarizes the progression of our understanding of HDL proteomic complexity and points out key experimental observations that reinforce the functional diversity of HDL. The possibility of specific HDL subspecies with distinct functions, the evidence supporting this concept, and some of the best examples of experimentally defined HDL subspecies are also discussed. Finally, key challenges facing the field are highlighted, particularly the need to identify and define the function of HDL subspecies to better inform attempts to pharmacologically manipulate HDL for the benefit of cardiovascular disease and possibly other maladies. Working at the Pasteur Institute in Paris in 1929, Michel Macheboeuf reported the first detailed isolation of a lipid-rich “α-globulin” from horse serum, a fraction that would later become known as high density lipoprotein (HDL) (1Macheboeuf M. Recherches sur les phosphoaminolipides du sérum sanguin. Nature des phospholipides liés aux albumines du sérum de Cheval à l’état de cenapses acido-précipitables.Bull. Soc. Chim. Biol. (Paris). 1929; 11: 485-503Google Scholar). Although relatively rapid progress was made at characterizing the lipid composition of this fraction - phospholipids (PL, ~25%), cholesterol (~4%), triglycerides (TG, ~3%) and cholesteryl esters (CE,~12%) - it took nearly 40 years to begin to sort out the complexities of the protein component. Part of the delay may have resulted from the widespread believe that “apo-HDL” was likely single protein entity, much like low-density lipoprotein (LDL), which was dominated by a single polypeptide later identified as apolipoprotein (apo)B. Using amino acid analyses, early electrophoresis methods, and a variety chromatographic techniques, the first HDL protein moiety was identified in the late 1960s. Variously referred to as α-protein, “R-Thr” peptide (2Shore B. Shore V. Heterogeneity in protein subunits of human serum high-density lipoproteins.Biochemistry. 1968; 7: 2773-2777Crossref PubMed Google Scholar), fraction III (3Scanu A. Toth J. Edelstein C. Koga S. Stiller E. Fractionation of human serum high density lipoprotein in urea solutions. Evidence for polypeptide heterogeneity.Biochemistry. 1969; 8: 3309-3316Crossref PubMed Google Scholar), and fraction II peptide (4Rudman D. Garcia L.A. Howard C.H. A new method for isolating the nonidentical protein subunits of human plasma alpha-lipoprotein.J. Clin. Invest. 1970; 49: 365-372Crossref PubMed Google Scholar), it later became known as apoA-I (5Kostner G. Alaupovic P. Studies of the composition and structure of plasma lipoproteins. C- and N-terminal amino acids of the two nonidentical polypeptides of human plasma apolipoprotein A.FEBS Lett. 1971; 15: 320-324Crossref PubMed Scopus (69) Google Scholar). Working in parallel, these laboratories also first recognized the possibility for heterogeneity within the HDL protein complement. This led to the identification of a second protein later known as apoA-II (5Kostner G. Alaupovic P. Studies of the composition and structure of plasma lipoproteins. C- and N-terminal amino acids of the two nonidentical polypeptides of human plasma apolipoprotein A.FEBS Lett. 1971; 15: 320-324Crossref PubMed Scopus (69) Google Scholar). The significance of this observation was not lost on these early pioneers. For example, the Shores observed, “The existence of multiple forms of polypeptides may be of considerable significance in the physiological and biochemical functions of the lipoproteins….” (6Shore B. Shore V. Isolation and characterization of polypeptides of human serum lipoproteins.Biochemistry. 1969; 8: 4510-4516Crossref PubMed Google Scholar). Around the same time, the Alupovic laboratory documented still further complexity in the HDL proteome by identifying the first of the minor constituents, the apoC peptides (5Kostner G. Alaupovic P. Studies of the composition and structure of plasma lipoproteins. C- and N-terminal amino acids of the two nonidentical polypeptides of human plasma apolipoprotein A.FEBS Lett. 1971; 15: 320-324Crossref PubMed Scopus (69) Google Scholar). Soon after, Mahley and colleagues noted the presence of the arginine-rich apoE in light fractions of HDL in dogs (HDL-1) (7Mahley R.W. Innerarity T.L. Interaction of canine and swine lipoproteins with the low density lipoprotein receptor of fibroblasts as correlated with heparin/manganese precipitability.J. Biol. Chem. 1977; 252: 3980-3986Abstract Full Text PDF PubMed Google Scholar), while others found apoD in the more dense HDL fractions (8Ayrault-Jarrier M. Alix J.F. Polonovski J. Presence and isolation of 2 lipoproteins immunologically related to apolipoprotein A I in human serum [article in French].Biochimie. 1980; 62: 51-59Crossref PubMed Scopus (0) Google Scholar). ApoF was also identified (9Olofsson S.O. McConathy W.J. Alaupovic P. Isolation and partial characterization of a new acidic apolipoprotein (apolipoprotein F) from high density lipoproteins of human plasma.Biochemistry. 1978; 17: 1032-1036Crossref PubMed Google Scholar). Improvements in SDS-PAGE technology in the early 1980s revealed serum amyloid A (SAA) (10Bausserman L.L. Herbert P.N. McAdam K.P. Heterogeneity of human serum amyloid A proteins.J. Exp. Med. 1980; 152: 641-656Crossref PubMed Google Scholar) and apoA-IV (11Vézina C.A. Milne R.W. Weech P.K. Marcel Y.L. Apolipoprotein distribution in human lipoproteins separated by polyacrylamide gradient gel electrophoresis.J. Lipid Res. 1988; 29: 573-585Abstract Full Text PDF PubMed Google Scholar) in human HDL. Immunological studies using antibodies raised against isolated HDL showed that paraoxonase 1 (PON1) coeluted with apoA-I upon gel filtration of plasma and that immunoabsorption of apoA-I removed 90% of PON1 from plasma (12Blatter M.C. James R.W. Messmer S. Barja F. Pometta D. Identification of a distinct human high-density lipoprotein subspecies defined by a lipoprotein-associated protein, K-45. Identity of K-45 with paraoxonase.Eur. J. Biochem. 1993; 211: 871-879Crossref PubMed Scopus (308) Google Scholar). Similarly, immunoabsorption of clustrin (apoJ) pulled down apoA-I, indicating its presence on HDL. It also became clear that several plasma enzymatic activities were associated with HDL, at least transiently. Single-spin vertical ultracentrifugation studies showed that lecithin:cholesterol acyl transferase (LCAT) was present in the higher density subfractions of HDL and also in LDL (13Campos E. McConathy W.J. Distribution of lipids and apolipoproteins in human plasma by vertical spin ultracentrifugation.Arch. Biochem. Biophys. 1986; 249: 455-463Crossref PubMed Scopus (8) Google Scholar). Other factors known to remodel HDL included cholesteryl ester transfer protein (CETP) (14Marcel Y.L. Vezina C. Teng B. Sniderman A. Transfer of cholesterol esters between human high density lipoproteins and triglyceride-rich lipoproteins controlled by a plasma protein factor.Atherosclerosis. 1980; 35: 127-133Abstract Full Text PDF PubMed Scopus (61) Google Scholar), phospholipid transfer protein (PLTP) (15Tall A.R. Krumholz S. Olivecrona T. Deckelbaum R.J. Plasma phospholipid transfer protein enhances transfer and exchange of phospholipids between very low density lipoproteins and high density lipoproteins during lipolysis.J. Lipid Res. 1985; 26: 842-851Abstract Full Text PDF PubMed Google Scholar), and platelet-activating factor aryl hydrolase (PAF-AH) (16Stafforini D.M. McIntyre T.M. Carter M.E. Prescott S.M. Human plasma platelet-activating factor acetylhydrolase. Association with lipoprotein particles and role in the degradation of platelet-activating factor.J. Biol. Chem. 1987; 262: 4215-4222Abstract Full Text PDF PubMed Google Scholar), also known as lipoprotein-associated phospholipase A2. By the early 1990s, HDL was generally thought to contain somewhere around 15 proteins. Indeed, one of the first two-dimensional gel electrophoresis studies of human plasma HDL referred to a set of “established HDL proteins” that included apoA-I, A-II, A-IV, C-II, C-III, D, and E, with mention of about 8 others that “can associate” with HDL (17James R.W. Hochstrasser D. Tissot J.D. Funk M. Appel R. Barja F. Pellegrini C. Muller A.F. Pometta D. Protein heterogeneity of lipoprotein particles containing apolipoprotein A-I without apolipoprotein A-II and apolipoprotein A-I with apolipoprotein A-II isolated from human plasma.J. Lipid Res. 1988; 29: 1557-1571Abstract Full Text PDF PubMed Google Scholar) (six other protein spots were observed but not identified). In general, these fell into four major functional groups: i) proteins associated with lipid transport or lipoprotein integrity, i.e., the “apos”ii) lipolytic enzymes, such as LCAT and PON; iii) lipid transfer proteins, such as CETP and PLTP; and iv) acute-phase response proteins, such as SAA and apoJ/clustrin. The presence of these proteins fit well into the general dogma of a primary HDL function as a lipid transport vehicle. Classical biochemical and immunological approaches were typically hampered by sensitivity issues or the fact that one needed a preconceived notion of what one was looking for prior to the experiment. However, the development of soft ionization techniques, such as electrospray (18Fenn J.B. Mann M. Meng C.K. Wong S.F. Whitehouse C.M. Electrospray ionization for mass spectrometry of large biomolecules.Science. 1989; 246: 64-71Crossref PubMed Google Scholar) and matrix-assisted laser desorption ionization (MALDI) (19Hillenkamp F. Karas M. Beavis R.C. Chait B.T. Matrix-assisted laser desorption/ionization mass spectrometry of biopolymers.Anal. Chem. 1991; 63: 1193A-1203ACrossref PubMed Google Scholar), for introducing large molecules into a mass spectrometer (MS) allowed for the development of sensitive and unbiased tandem MS approaches capable of identifying components of complex protein mixtures. In general, ionized proteolytic peptides in the gas phase are sorted by mass and then subjected to a controlled fragmentation, typically cleaving at the peptide bonds. The fragments are then evaluated by a second mass analyzer, and the resulting patterns are bioinformatically compared with theoretical patterns for all known proteins, effectively sequencing the peptides and producing a redundant list of proteins present in the initial mixture. Given that modern MS instrumentation can identify proteins across a 10,000-fold difference in concentration with sensitivities down to the nano- to picomole range, it is not uncommon to identify hundreds of proteins in a given biological sample. Although strategies can vary significantly between laboratories, MS-based proteomic approaches applied to HDL fall into two rough categories. In the first, HDL proteins are first separated by gel electrophoresis either by size only (1D) or by charge and then size (2D). The resulting gel spots are excised, digested with a protease, such as trypsin, and then identified by tandem MS. The second approach, sometimes referred to as the “shotgun” technique, starts by trypsinizing all proteins together in solution. The peptides are then separated by HPLC (typically by reverse phase) and subjected to electrospray ionization (ESI) as they elute from the column and then analyzed by tandem MS. A more detailed description of proteomic technologies can be found in Ref. 20Cañas B. López-Ferrer D. Ramos-Fernández A. Camafeita E. Calvo E. Mass spectrometry technologies for proteomics.Brief Funct. Genomic Proteomic. 2006; 4: 295-320Crossref PubMed Scopus (0) Google Scholar. One of the first applications of these technologies toward the HDL proteome was pursued by Karlsson et al. (21Karlsson H. Leanderson P. Tagesson C. Lindahl M. Lipoproteomics II: mapping of proteins in high-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry.Proteomics. 2005; 5: 1431-1445Crossref PubMed Scopus (143) Google Scholar). Using a 2D electrophoresis/tandem MS approach, they identified 13 proteins in HDL2 and HDL3 separated by density ultracentrifugation from healthy donors. Of these, 11 were previously suspected HDL components from the biochemical studies summarized above. In addition, this study highlighted a key advantage of the charge dimension of the electrophoretic approach in that multiple isoforms of apoA-I, apoA-II, apoC-III, apoE, apoM, SAA, and SAA-4 were identified that varied with respect to sequence differences or posttranslational modifications. The next year, Rezaee et al. (22Rezaee F. Casetta B. Levels J.H. Speijer D. Meijers J.C. Proteomic analysis of high-density lipoprotein.Proteomics. 2006; 6: 721-730Crossref PubMed Scopus (150) Google Scholar) used a multipronged approach of 1D and 2D electrophoresis, shotgun proteomics, and immunological assays to study centrifugally isolated total HDL from normal donors. This study first identified key mediators of the complement system - C3, C1 inhibitor, and complement factor H - implicating HDL in innate immunity. Likely, the most high-profile proteomic analysis of HDL was performed by Vaisar et al. (23Vaisar T. Pennathur S. Green P.S. Gharib S.A. Hoofnagle A.N. Cheung M.C. Byun J. Vuletic S. Kassim S. Singh P. et al.Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL.J. Clin. Invest. 2007; 117: 746-756Crossref PubMed Scopus (727) Google Scholar). Using centrifugally isolated total HDL or HDL3 from normal subjects, they identified 48 HDL proteins. These included additional members of the complement family, strengthening the argument for a role of HDL in innate immunity. Others, such as the serine protease inhibitor (SERPIN) family, showed a clear theme of protease inhibition, including those involved in hemostasis. Overall, the Rezaee and Vaisar studies clearly demonstrated that the proteins associated with HDL are not limited to those involved in lipid transport. These concepts drove tremendous interest in further exploring the HDL proteome. By the end of 2012, several laboratories had published some 14 studies that applied various MS techniques to understand the human HDL proteome. As sensitivity of the instrumentation has increased, so has the number of proteins that are proposed to be associated with HDL. These studies included HDL samples isolated by traditional density ultracentrifugation (21Karlsson H. Leanderson P. Tagesson C. Lindahl M. Lipoproteomics II: mapping of proteins in high-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry.Proteomics. 2005; 5: 1431-1445Crossref PubMed Scopus (143) Google Scholar, 22Rezaee F. Casetta B. Levels J.H. Speijer D. Meijers J.C. Proteomic analysis of high-density lipoprotein.Proteomics. 2006; 6: 721-730Crossref PubMed Scopus (150) Google Scholar, 23Vaisar T. Pennathur S. Green P.S. Gharib S.A. Hoofnagle A.N. Cheung M.C. Byun J. Vuletic S. Kassim S. Singh P. et al.Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL.J. Clin. Invest. 2007; 117: 746-756Crossref PubMed Scopus (727) Google Scholar, 24Heller M. Stalder D. Schlappritzi E. Hayn G. Matter U. Haeberli A. Mass spectrometry-based analytical tools for the molecular protein characterization of human plasma lipoproteins.Proteomics. 2005; 5: 2619-2630Crossref PubMed Scopus (63) Google Scholar, 25Hortin G.L. Shen R.F. Martin B.M. Remaley A.T. Diverse range of small peptides associated with high-density lipoprotein.Biochem. Biophys. Res. Commun. 2006; 340: 909-915Crossref PubMed Scopus (52) Google Scholar, 26Davidson W.S. Silva R.A. Chantepie S. Lagor W.R. Chapman M.J. Kontush A. Proteomic analysis of defined HDL subpopulations reveals particle-specific protein clusters: relevance to antioxidative function.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 870-876Crossref PubMed Scopus (284) Google Scholar, 27Alwaili K. Bailey D. Awan Z. Bailey S.D. Ruel I. Hafiane A. Krimbou L. Laboissiere S. Genest J. The HDL proteome in acute coronary syndromes shifts to an inflammatory profile.Biochim. Biophys. Acta. 2012; 1821: 405-415Crossref PubMed Scopus (139) Google Scholar, 28Holzer M. Wolf P. Curcic S. Birner-Gruenberger R. Weger W. Inzinger M. El-Gamal D. Wadsack C. Heinemann A. Marsche G. Psoriasis alters HDL composition and cholesterol efflux capacity.J. Lipid Res. 2012; 53: 1618-1624Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 29Holzer M. Birner-Gruenberger R. Stojakovic T. El-Gamal D. Binder V. Wadsack C. Heinemann A. Marsche G. Uremia alters HDL composition and function.J. Am. Soc. Nephrol. 2011; 22: 1631-1641Crossref PubMed Scopus (187) Google Scholar, 30Weichhart T. Kopecky C. Kubicek M. Haidinger M. Doller D. Katholnig K. Suarna C. Eller P. Tolle M. Gerner C. et al.Serum amyloid A in uremic HDL promotes inflammation.J. Am. Soc. Nephrol. 2012; 23: 934-947Crossref PubMed Scopus (153) Google Scholar, 31Mangé A. Goux A. Badiou S. Patrier L. Canaud B. Maudelonde T. Cristol J.P. Solassol J. HDL proteome in hemodialysis patients: a quantitative nanoflow liquid chromatography-tandem mass spectrometry approach.PLoS ONE. 2012; 7: e34107Crossref PubMed Scopus (56) Google Scholar), immunoaffinity capture (32Watanabe J. Charles-Schoeman C. Miao Y. Elashoff D. Lee Y.Y. Katselis G. Lee T.D. Reddy S.T. Proteomic profiling following immunoaffinity capture of high-density lipoprotein: association of acute-phase proteins and complement factors with proinflammatory high-density lipoprotein in rheumatoid arthritis.Arthritis Rheum. 2012; 64: 1828-1837Crossref PubMed Scopus (109) Google Scholar), size exclusion chromatography (33Gordon S.M. Deng J. Lu L.J. Davidson W.S. Proteomic characterization of human plasma high density lipoprotein fractionated by gel filtration chromatography.J. Proteome Res. 2010; 9: 5239-5249Crossref PubMed Scopus (148) Google Scholar), and even anion-exchange and isoelectric focusing (S. M. Gordon and W. S. Davidson, unpublished observations). A summary of the key experimental details for each of these studies is given in Table 1. As a result of differences in instrumentation, sensitivity, HDL isolation technique, patient donors, and protein identification algorithms, the total list of putative HDL proteins can vary dramatically from study to study. Several estimates of the total number of HDL proteins have reached into the hundreds and have included hits that may induce skepticism. For example, certain intracellular and cell-surface proteins and even human skin keratin have been detected in HDL preparations. In an effort to manage this large amount of information and attempt to filter out instrument- or laboratory-specific artifacts, we have initiated the HDL Proteome Watch (http://homepages.uc.edu/~davidswm/HDLproteome.html or http://www.hdlforum.org/resources/links). This project tracks published reports that have used modern MS methodologies to study human HDL samples that have been physically separated in some way from plasma or serum. Currently, 204 individual proteins have been detected in human HDL samples. Of these, 85 proteins have appeared in at least three different studies (from independent laboratories), representing the best current estimate of the HDL proteome. These proteins are listed in Fig. 1.TABLE 1Experimental details of recent MS-based proteomic studies of HDLReferencePatient PopulationHDL Type and Separation TechniqueMS ApproachMS Database SearchedGel Separation?(21Karlsson H. Leanderson P. Tagesson C. Lindahl M. Lipoproteomics II: mapping of proteins in high-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry.Proteomics. 2005; 5: 1431-1445Crossref PubMed Scopus (143) Google Scholar)Pooled plasma from healthy adults (n = 4)HDL2, HDL3 isolated by density gradient UC (KBr)MALDI-TOFNCBI and SwissProtYes2D gel(24Heller M. Stalder D. Schlappritzi E. Hayn G. Matter U. Haeberli A. Mass spectrometry-based analytical tools for the molecular protein characterization of human plasma lipoproteins.Proteomics. 2005; 5: 2619-2630Crossref PubMed Scopus (63) Google Scholar)Pool of plasma from >10,000 donorsTotal HDL isolated by one-step density gradient UC (KBr)MALDI-TOFUniProtYes2D gel(25Hortin G.L. Shen R.F. Martin B.M. Remaley A.T. Diverse range of small peptides associated with high-density lipoprotein.Biochem. Biophys. Res. Commun. 2006; 340: 909-915Crossref PubMed Scopus (52) Google Scholar)1 healthy donorTotal HDL isolated by density gradient UC (KBr)HPLC to separate peptides MALDI- TOFDetails not givenNo(22Rezaee F. Casetta B. Levels J.H. Speijer D. Meijers J.C. Proteomic analysis of high-density lipoprotein.Proteomics. 2006; 6: 721-730Crossref PubMed Scopus (150) Google Scholar)Healthy donors (n not reported)Total HDL isolated by one-step density gradient UC (KBr) and immunoisolationMALDI-TOF, isotope coded affinity tag plus Western blot analysisDetails not givenYes1D and 2D gel(23Vaisar T. Pennathur S. Green P.S. Gharib S.A. Hoofnagle A.N. Cheung M.C. Byun J. Vuletic S. Kassim S. Singh P. et al.Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL.J. Clin. Invest. 2007; 117: 746-756Crossref PubMed Scopus (727) Google Scholar)Studies of total HDL: 20 males; studies of HDL3: 6 healthy males and 7 with CADTotal HDL and HDL3 isolated by density gradient UC (KBr)LC-ESI-MS/MSInternational Protein IndexNo(26Davidson W.S. Silva R.A. Chantepie S. Lagor W.R. Chapman M.J. Kontush A. Proteomic analysis of defined HDL subpopulations reveals particle-specific protein clusters: relevance to antioxidative function.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 870-876Crossref PubMed Scopus (284) Google Scholar)9 healthy normolipidemic males and 3 samples, each consisting of a pool from 20 healthy normolipidemic malesHDL2b, 2a, 3a, 3b and 3c isolated by density gradient UC (KBr)LC-ESI-MS/MSSwissProtNo(33Gordon S.M. Deng J. Lu L.J. Davidson W.S. Proteomic characterization of human plasma high density lipoprotein fractionated by gel filtration chromatography.J. Proteome Res. 2010; 9: 5239-5249Crossref PubMed Scopus (148) Google Scholar)3 healthy normolipidemic males“HDL” isolated by high resolution size exclusion chromatography followed by lipid removal agentLC-ESI MS/MSUniProt and SwissProtNo(27Alwaili K. Bailey D. Awan Z. Bailey S.D. Ruel I. Hafiane A. Krimbou L. Laboissiere S. Genest J. The HDL proteome in acute coronary syndromes shifts to an inflammatory profile.Biochim. Biophys. Acta. 2012; 1821: 405-415Crossref PubMed Scopus (139) Google Scholar)10 healthy adults, 10 with stable coronary disease and 10 with acute coronary syndrome. Age-matched malesTotal HDL separated by density gradient UC (KBr)LC-ESI MS/MSUniProtYes1D gel(32Watanabe J. Charles-Schoeman C. Miao Y. Elashoff D. Lee Y.Y. Katselis G. Lee T.D. Reddy S.T. Proteomic profiling following immunoaffinity capture of high-density lipoprotein: association of acute-phase proteins and complement factors with proinflammatory high-density lipoprotein in rheumatoid arthritis.Arthritis Rheum. 2012; 64: 1828-1837Crossref PubMed Scopus (109) Google Scholar)Aged-matched females with rheumatoid arthritis: 4 with anti-inflammatory HDL index, and 4 with proinflammatory HDL indexTotal HDL isolated by immunoaffinity captureLC-ESI MS/MSUniProtYesIEF gel(28Holzer M. Wolf P. Curcic S. Birner-Gruenberger R. Weger W. Inzinger M. El-Gamal D. Wadsack C. Heinemann A. Marsche G. Psoriasis alters HDL composition and cholesterol efflux capacity.J. Lipid Res. 2012; 53: 1618-1624Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar)19 healthy adults and 27 with end stage renal disease on hemodialysis. Males and femalesTotal HDL isolated by one-step density gradient UC (KBr)LC-ESI-MS/MSNCBINo(29Holzer M. Birner-Gruenberger R. Stojakovic T. El-Gamal D. Binder V. Wadsack C. Heinemann A. Marsche G. Uremia alters HDL composition and function.J. Am. Soc. Nephrol. 2011; 22: 1631-1641Crossref PubMed Scopus (187) Google Scholar)15 healthy adults and 15 with psoriasis. Males and femalesTotal HDL isolated by two-step density gradient UC (KBr)LC-ESI-MS/MSSwissProtNo(30Weichhart T. Kopecky C. Kubicek M. Haidinger M. Doller D. Katholnig K. Suarna C. Eller P. Tolle M. Gerner C. et al.Serum amyloid A in uremic HDL promotes inflammation.J. Am. Soc. Nephrol. 2012; 23: 934-947Crossref PubMed Scopus (153) Google Scholar)10 healthy adults and 10 with end stage renal disease. Males and femalesTotal HDL isolated by density gradient UC (KBr)LC-ESI-MS/MSSwissProtNo(31Mangé A. Goux A. Badiou S. Patrier L. Canaud B. Maudelonde T. Cristol J.P. Solassol J. HDL proteome in hemodialysis patients: a quantitative nanoflow liquid chromatography-tandem mass spectrometry approach.PLoS ONE. 2012; 7: e34107Crossref PubMed Scopus (56) Google Scholar)30 healthy adults and 30 with end stage kidney disease on hemodialysis. Males and femalesTotal HDL isolated by three-step density gradient UC (KBr)MALDI-TOF and iTRAQ labeling prior to LC-ESI MS/MSUniProtYesIEF gelIEF, isoelectric focusing; iTRAQ, multiplexed isobaric-tagged reagents produced by Sciex; UC, ultracentrifugation. Open table in a new tab IEF, isoelectric focusing; iTRAQ, multiplexed isobaric-tagged reagents produced by Sciex; UC, ultracentrifugation. Figure 2 shows a gene ontology enrichment analysis showing the general functional classifications of the consensus proteins listed in Fig. 1. Although many HDL proteins fall within the general area of lipid metabolism, proteins with numerous other functions are also present, including proteins involved in hemostasis, such as fibrinogen, and several of the SERPINs involved in the clotting cascade. There are a striking number of HDL proteins involved in the inflammatory/immune response, including numerous members of the complement system and its associated proteolysis inhibitors, apoJ, and vitronectin. Also clearly represented are acute-phase response proteins, such as SAA and lipopolysaccharide (LPS)-binding protein (LPB). Surprisingly, there are also proteins involved in heme and iron metabolism, such as hemoglobin, transferrin, and hemopexin, as well as those with a host of additional and enigmatic functions, ranging from platelet regulation to vitamin binding and transport. HDL is most widely recognized for its ability to shuttle cholesterol from the periphery to the liver for catabolism/excretion during the process of reverse cholesterol transport (RCT). Numerous studies have shown that HDL and most of its apolipoproteins can promote lipid efflux from cells via a number of mechanisms (34Adorni M.P. Zimetti F. Billheimer J.T. Wang N. Rader D.J. Phillips M.C. Rothblat G.H. The roles of different pathways in the release of cholesterol from macrophages.J. Lipid Res. 2007; 48: 2453-2462Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar) and can deliver cholesteryl esters to the liver in the process of selective uptake (35Gu X. Trigatti B. Xu S. Acton S. Babitt J. Krieger M. The efficient cellular uptake of high density lipoprotein lipids via scavenger receptor class B type I requires not only receptor-mediated surface binding but also receptor-specific lipid transfer mediated by its extracellular domain.J. Biol. Chem. 1998; 273 ([Erratum. 1998. J. Biol. Chem. 273: 35388.]): 26338-26348Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). The importance of this process has been demonstrated by in vivo models of RCT showing that genetic lowering of plasma HDL decreases the appearance of macrophage-derived cholesterol in the feces (36Moore R.E. Navab M. Millar J.S. Zimetti F. Hama S. Rothblat G.H. Rader D.J. Increased atherosclerosis in mice lacking apolipoprotein A