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 HDL2/3 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 α-1-antitrypsin, α-2-HS-glycoprotein, serotransferrin, apolipoprotein A-IV and others, are not associated with HDL2/3 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 α-1-antitrypsin. We propose that this refined method might become a standard in proteomic assessment of HDL2/3 making data from clinical cohorts more comparable and reproducible.
Highlights
“highly purified” fractions of HDL2/3 to provide a reliable and accurate analysis of the HDL proteome for biomarker discovery
We used longer centrifugation tubes (76 mm), which allowed us for the complete removal of all apoB-containing lipoproteins within one ultracentrifugation step
The proteomic analysis revealed that many proteins which have recently been described to be associated with HDL2/3, including α-1-antitrypsin, α-2-HS-glycoprotein, serotransferrin, apoA-IV are exclusively associated with the fraction containing lipid-poor apoA-I, human serum albumin (HSA) and potentially other contaminants (Table 1)
Summary
Molecular characterization of “highly purified” fractions of HDL. We established a refined strategy to isolate “highly purified” HDL2/3 for proteomic characterization. HDL isolated by ultracentrifugation was further purified by size using either native gel electrophoresis or size exclusion chromatography (Fig. 1). This methodology has the advantage that contaminants that overlap in density can be removed by separation in size. Our analysis indicated that HDL isolated by ultracentrifugation without additional purification contained 26 different proteins (Table 1) This rather low number of identified proteins is a result of our improved isolation procedure by using a one-step ultracentrifugation in combination with longer centrifugation tubes (Supplemental Fig. 1). The proteomic analysis revealed that many proteins which have recently been described to be associated with HDL2/3, including α-1-antitrypsin, α-2-HS-glycoprotein, serotransferrin, apoA-IV are exclusively associated with the fraction containing lipid-poor apoA-I, HSA and potentially other contaminants (Table 1)
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