Mass spectrometric determination of apolipoprotein molecular stoichiometry in reconstituted high density lipoprotein particles

Abstract

Plasma HDL-cholesterol and apolipoprotein A-I (apoA-I) levels are strongly inversely associated with cardiovascular disease. However, the structure and protein composition of HDL particles is complex, as native and synthetic discoidal and spherical HDL particles can have from two to five apoA-I molecules per particle. To fully understand structure-function relationships of HDL, a method is required that is capable of directly determining the number of apolipoprotein molecules in heterogeneous HDL particles. Chemical cross-linking followed by SDS polyacrylamide gradient gel electrophoresis has been previously used to determine apolipoprotein stoichiometry in HDL particles. However, this method yields ambiguous results due to effects of cross-linking on protein conformation and, subsequently, its migration pattern on the gel. Here, we describe a new method based on cross-linking chemistry followed by MALDI mass spectrometry that determines the absolute mass of the cross-linked complex, thereby correctly determining the number of apolipoprotein molecules in a given HDL particle. Using well-defined, homogeneous, reconstituted apoA-I-containing HDL, apoA-IV-containing HDL, as well as apoA-I/apoA-II-containing HDL, we have validated this method. The method has the capability to determine the molecular ratio and molecular composition of apolipoprotein molecules in complex reconstituted HDL

Severe hypoalphalipoproteinaemia in a child with acute post-streptococcal glomerulonephritis (APSGN)

“…This has been demonstrated in experimental models of nephrotic syndrome where high-density lipoproteinuria has been implicated in the pathogenesis of dyslipidemia 7. The preferential loss of Apo A1 in the urine might be explained by its relatively low molecular mass of 28 kDa compared to that of albumin which is 67 kDa 8. Our patient had a low Apo A1 of 0.63 g/L.…”

Apolipoprotein A-II-mediated Conformational Changes of Apolipoprotein A-I in Discoidal High Density Lipoproteins

“…The peak originating from cross-linked dimeric apoA-I in A-I dHDL and A-I/A-II dHDL showed comparable mass shifts upon cross-linking (3,760 and 3,940 Da, respectively). These mass shifts are representative of the average addition of 27 and 28 BS 3 cross-links, respectively (29). Hence, the outcome suggested an overall preservation of "double-belt" architecture for apoA-I in mixed particles, as for apoA-I in A-I dHDL (36,37,44,45).…”

“…2A, peak 1) and ϳ59.9 kDa ( Fig. 2A, peak 2), originating from cross-linked monomeric and dimeric apoA-I, respectively (29). The mass ϳ59.9 kDa, the highest molecular weight appearing in the spectrum represents the mass of two apoA-I molecules cross-linked together.…”

“…MALDI-MS-Mass spectrometry measurements were carried out using methods developed in our laboratory as reported before (12,29). Briefly, the cross-linker, bis(sulfosuccinimidyl-)suberate (BS 3 , Pierce), was added to dHDL particles at a total protein to cross-linker molar ratio of 1:100 and the solution was incubated for 24 h. Upon delipidation, samples were subjected to mass spectrometry measurements on a Bruker Daltonics MALDI-TOF mass spectrometer (Billerica, MA) (12,29).…”

“…This indicated the presence of near neighbor interactions between apoA-I and apoA-II. Low intensity of the ϳ69.5 kDa peak in the MALDI-MS spectrum could be simply due to the nonquantitative nature of MALDI-MS (29), or due to the specific ion suppression of this mass (38,39). The strong evidence available for the presence of a trimer, but not apoA-I-apoA-II heterodimer (Figs.…”