Separation and characterization of human high-density apolipoproteins using a nonaqueous modifier in capillary electrophoresis-mass spectrometry.
The separation and characterization of human apolipoproteins and their isoforms was investigated using capillary electrophoresis (CE) in combination with mass spectrometry (MS). The focus of these analyses was the major protein constituents of plasma high-density lipoproteins, apolipoprotein A-I and A-II. Using aqueous buffers in CE, no separation between apolipoprotein A-I and A-II was observed. With the addition of 10-20% acetonitrile, however, the two species could be separated. Furthermore, multiple peaks for each of the apolipoprotein species were observed under these CE conditions. In order to identify and characterize the components, these separations were then coupled with online mass spectrometric detection (CE-MS). Our CE-MS results suggest that the multiple components observed in the acetonitrile-containing CE separation appear to be oxidized forms of the proteins in addition to native forms of the apolipoprotein A-I and A-II. These data are in agreement with previous reports that the methionine residues of the high-density lipoproteins (HDLs) are sensitive to oxidation, which in turn, alters their lipid binding characteristics and secondary structure. In addition to oxidized forms of the proteins, apolipoprotein A-II contained additional components, which varied in mass by 128 Da. The structural differences between these components were determined by proteolytic digestion and tandem MS. Using these techniques, we determined that these components were due to truncation of the C-terminal glutamine amino acid residue on apolipoprotein A-II. These results demonstrate that CE in combination with MS is a promising technique for screening and characterizing isomers of plasma apolipoproteins.
Deterding
Leesa J
LJ
Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Cutalo
Jenny M
JM
Khaledi
Morteza
M
Tomer
Kenneth B
KB
D000097:Acetonitriles / Q000737:chemistry*; D016632:Apolipoprotein A-I / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D016633:Apolipoprotein A-II / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D001053:Apolipoproteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D003324:Coronary Artery Disease / Q000175:diagnosis; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000295:instrumentation; D012307:Risk Factors; D020539:Sequence Analysis, Protein; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D000097:Acetonitriles; D016632:Apolipoprotein A-I; D016633:Apolipoprotein A-II; D001053:Apolipoproteins; C032159:acetonitrile