title: Influence of HSA and IgG on LDL oxidation studied by size-exclusion chromatography and phospholipid profiling using MALDI tandem-mass spectrometry.
abstract: In the present study a direct detection approach combining size-exclusion chromatography (SEC) and matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight tandem-mass spectrometry (MALDI-QIT-TOF-MS/MS) was applied to investigate the influence of HSA and IgG on LDL oxidation in vitro. SEC analysis showed an increase of protein aggregation during LDL-oxidation that could be essentially suppressed in the presence of HSA. In parallel, lipid peroxidation measured by TBARS assay over 24h was inhibited by 95-100% in the presence of HSA but only 0-34% by IgG, respectively. MALDI phospholipid profiles showed considerable decrease of signals from PCs containing sn-2 PUFAs (18:2 or 20:4) accompanied by increase of sn-2 LPCs indicating for specific breakdown of PUFA-containing PLs during LDL-oxidation. These effects were nearly 100% inhibited in the presence of HSA but not by IgG, respectively. Among known pro-atherogenic PL species present in human plasma sphingomyelin (SM16:0) was bound in significant amounts to HSA but not IgG after incubation with oxLDL. Moreover, our investigation showed that LPCs containing SAFAs (16:0 or 18:0) were specifically bound to HSA, while those containing PUFAs (18:2 and 18:3) were preferentially associated with IgG. In summary, the presented methodology provides a promising platform for studying lipid-protein interactions in vivo.
authors.affiliation: Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria. gerald.stubiger@meduniwien.ac.at
authors.forename: Gerald
authors.initials: G
authors.lastname: Stübiger
authors.forename: Grazyna
authors.initials: G
authors.lastname: Sobal
authors.forename: Kurt
authors.initials: K
authors.lastname: Widhalm
authors.forename: Omar
authors.initials: O
authors.lastname: Belgacem
authors.forename: Katharina
authors.initials: K
authors.lastname: Pock
mesh_terms: D000328:Adult; D050197:Atherosclerosis; D002850:Chromatography, Gel / Q000379:methods*; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification / Q000494:pharmacology*; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008244:Lysophosphatidylcholines / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D010084:Oxidation-Reduction / Q000187:drug effects; D010743:Phospholipids / Q000378:metabolism*; D012709:Serum Albumin / Q000302:isolation & purification / Q000494:pharmacology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013109:Sphingomyelins / Q000378:metabolism
chemical_list: D005231:Fatty Acids, Unsaturated; D007074:Immunoglobulin G; D008077:Lipoproteins, LDL; D008244:Lysophosphatidylcholines; D010743:Phospholipids; D012709:Serum Albumin; D013109:Sphingomyelins; C099252:oxidized low density lipoprotein