Proteomics and lipids of lipoproteins isolated at low salt concentrations in D2O/sucrose or in KBr.
There is much interest in the significance of apolipoproteins and proteins that are noncovalently associated with lipoproteins. It is possible that the high ionic strength used for isolation of lipoproteins with KBr and NaI could alter the pattern of associated exchangeable proteins. Here we describe lipoprotein classes fractionation from up to 0.5 ml of serum or plasma with buffers of physiological ionic strength and pH prepared with deuterium oxide (D(2)O) and sucrose. An advantage of the D(2)O/sucrose procedure was that the lipoproteins could be directly analyzed by the techniques described without need for desalting. We compared the isolated lipoproteins with those obtained using ultracentrifugation in KBr from the same plasma pool. Electrophoretic homogeneity of the lipoproteins was very similar using the two methods, as well as their lipid composition evaluated by HPLC. Two-dimensional electrophoresis and surface-enhanced laser adsorption/ionization time-of-flight mass spectrometry indicated that the patterns of exchangeable proteins of VLDL isolated using with the two procedures were very similar. However, significant differences were found in the profiles of LDL and HDL, indicating that the D(2)O/sucrose method allowed a more complete characterization of its exchangeable apolipoproteins and proteins.
Ståhlman
Marcus
M
AstraZeneca R&D, Mölndal, Sweden.
Davidsson
Pia
P
Kanmert
Ida
I
Rosengren
Birgitta
B
Borén
Jan
J
Fagerberg
Björn
B
Camejo
Germán
G
D001053:Apolipoproteins / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification; D001965:Bromides; D017666:Deuterium Oxide; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006801:Humans; D008055:Lipids / Q000097:blood / Q000737:chemistry*; D008074:Lipoproteins / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification*; D017680:Potassium Compounds; D040901:Proteomics / Q000379:methods*; D012965:Sodium Chloride; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013395:Sucrose; D014461:Ultracentrifugation / Q000379:methods
D001053:Apolipoproteins; D001965:Bromides; D008055:Lipids; D008074:Lipoproteins; D017680:Potassium Compounds; D012965:Sodium Chloride; D013395:Sucrose; D017666:Deuterium Oxide; C039004:potassium bromide