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Refined purification strategy for reliable proteomic profiling of HDL_2/3: Impact on proteomic complexity.

ABSTRACT: Proteomics have extended the list of high-density lipoprotein (HDL) associated proteins to about 90. One of the major issues of global protein characterization is establishing specificity of association as opposed to contamination, a fact which has never been addressed for isolated HDL. We have developed a refined purification strategy to isolate HDL by density, followed by purification by size to generate "highly purified" fractions of HDL_2/3, which allow the reliable quantification of the HDL proteome for biomarker discovery. Mass spectrometry analysis revealed that the proteome of HDL_2/3 is composed of 10-16 different proteins, which is in striking contrast to previous reports. Importantly, proteomic analysis revealed that many proteins which have recently been described to be associated with HDL, including ?-1-antitrypsin, ?-2-HS-glycoprotein, serotransferrin, apolipoprotein A-IV and others, are not associated with HDL_2/3 and are exclusively found in a different molecular weight fraction containing human serum albumin, lipid-poor apolipoprotein A-I and other proteins. Interestingly, proteins found in this lower molecular weight fraction commonly share lipid-binding properties and enrichment of serum with free fatty acids/lysophophatidylcholine led to a significant increase in co-isolation of lipid-binding proteins such as albumin and ?-1-antitrypsin. We propose that this refined method might become a standard in proteomic assessment of HDL_2/3 making data from clinical cohorts more comparable and reproducible.

SUBMITTER: Holzer M

PROVIDER: S-EPMC5137140 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Refined purification strategy for reliable proteomic profiling of HDL<sub>2/3</sub>: Impact on proteomic complexity.

Holzer Michael M Kern Sabine S Birner-Grünberger Ruth R Curcic Sanja S Heinemann Akos A Marsche Gunther G

Scientific reports 20161205

Proteomics have extended the list of high-density lipoprotein (HDL) associated proteins to about 90. One of the major issues of global protein characterization is establishing specificity of association as opposed to contamination, a fact which has never been addressed for isolated HDL. We have developed a refined purification strategy to isolate HDL by density, followed by purification by size to generate "highly purified" fractions of HDL<sub>2/3</sub>, which allow the reliable quantification ...[more]

PMID: 27917957

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Project description:Recent clinical trials reported that increasing high-density lipoprotein-cholesterol (HDL-C) levels does not improve cardiovascular outcomes. We hypothesize that HDL proteome dynamics determine HDL cardioprotective functions. In this study, we characterized proteome profiles in HDL subclasses and established their functional connection. Mouse plasma was fractionized by fast protein liquid chromatography, examined for protein, cholesterial, phospholipid and trigliceride content. Small, medium and large (S/M/L)-HDL subclasseses were collected for proteomic analysis by mass spectrometry. Fifty-one HDL proteins (39 in S-HDL, 27 in M-HDL and 29 in L-HDL) were identified and grouped into 4 functional categories (lipid metabolism, immune response, coagulation, and others). Eleven HDL common proteins were identified in all HDL subclasses. Sixteen, 3 and 7 proteins were found only in S-HDL, M-HDL and L-HDL, respectively. We established HDL protein dynamic distribution in S/M/L-HDL and developed a model of protein composition change during HDL maturation. We found that cholesterol efflux and immune response are essential functions for all HDL particles, and amino acid metabolism is a special function of S-HDL, whereas anti-coagulation is special for M-HDL. Pon1 is recruited into M/L-HDL to provide its antioxidative function. ApoE is incorporated into L-HDL to optimize its cholesterial clearance function. Next, we acquired HDL proteome data from Pubmed and identified 12 replicated proteins in human and mouse HDL particle. Finally, we extracted 3 shared top moleccular pathways (LXR/RXR, FXR/RXR and acute phase response) for all HDL particles and 5 top disease/bio-functions differentially related to S/M/L-HDL subclasses, and presented one top net works for each HDL subclass. We conclude that beside their essencial functions of cholesterol efflux and immune response, HDL aquired antioxidative and cholesterol clearance functions by recruiting Pon1 and ApoE during HDL maturation.

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Refined purification strategy for reliable proteomic profiling of HDL_2/3: Impact on proteomic complexity.

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Refined purification strategy for reliable proteomic profiling of HDL<sub>2/3</sub>: Impact on proteomic complexity.

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Refined purification strategy for reliable proteomic profiling of HDL2/3: Impact on proteomic complexity.

Publications

Refined purification strategy for reliable proteomic profiling of HDL<sub>2/3</sub>: Impact on proteomic complexity.

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OmicsDI is part of the ELIXIR infrastructure

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Refined purification strategy for reliable proteomic profiling of HDL_2/3: Impact on proteomic complexity.