title: Refined purification strategy for reliable proteomic profiling of HDL2/3: 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 HDL2/3, which allow the reliable quantification of the HDL proteome for biomarker discovery. Mass spectrometry analysis revealed that the proteome of HDL2/3is 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 α-1-antitrypsin, α-2-HS-glycoprotein, serotransferrin, apolipoprotein A-IV and others, are not associated with HDL2/3and 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 α-1-antitrypsin. We propose that this refined method might become a standard in proteomic assessment of HDL2/3making data from clinical cohorts more comparable and reproducible.
authors.affiliation: Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Austria.
authors.forename: Michael
authors.initials: M
authors.lastname: Holzer
authors.affiliation: Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Austria.
authors.forename: Sabine
authors.initials: S
authors.lastname: Kern
authors.affiliation: Institute of Pathology and Proteomics Core Facility, Center for Medical Research, Medical University of Graz, Austria.
authors.forename: Ruth
authors.initials: R
authors.lastname: Birner-Grünberger
authors.affiliation: Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Austria.
authors.forename: Sanja
authors.initials: S
authors.lastname: Curcic
authors.affiliation: Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Austria.
authors.forename: Akos
authors.initials: A
authors.lastname: Heinemann
authors.affiliation: Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Austria.
authors.forename: Gunther
authors.initials: G
authors.lastname: Marsche
mesh_terms: D003432:Cross-Linking Reagents / Q000737:chemistry; D005227:Fatty Acids / Q000097:blood; D006801:Humans; D015151:Immunoblotting; D008075:Lipoproteins, HDL / Q000302:isolation & purification*; D054467:Phospholipases A2 / Q000378:metabolism; D010743:Phospholipids / Q000097:blood; D020543:Proteome / Q000378:metabolism; D040901:Proteomics / Q000379:methods*
chemical_list: D003432:Cross-Linking Reagents; D005227:Fatty Acids; D008075:Lipoproteins, HDL; D010743:Phospholipids; D020543:Proteome; D054467:Phospholipases A2