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 (182 or 204) 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 (SM160) was bound in significant amounts to HSA but not IgG after incubation with oxLDL. Moreover, our investigation showed that LPCs containing SAFAs (160 or 180) were specifically bound to HSA, while those containing PUFAs (182 and 183) were preferentially associated with IgG. In summary, the presented methodology provides a promising platform for studying lipid-protein interactions in vivo.