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A Phospholipidomic Analysis of All Defined Human Plasma Lipoproteins

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A Phospholipidomic Analysis of All Defined Human Plasma Lipoproteins

Monireh Dashti et al. Sci Rep.

Abstract

Since plasma lipoproteins contain both protein and phospholipid components, either may be involved in processes such as atherosclerosis. In this study the identification of plasma lipoprotein-associated phospholipids, which is essential for understanding these processes at the molecular level, are performed. LC-ESI/MS, LC-ESI-MS/MS and High Performance Thin Layer Chromatography (HPTLC) analysis of different lipoprotein fractions collected from pooled plasma revealed the presence of phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyeline (SM) only on lipoproteins and phosphatidylcholine (PC), Lyso-PC on both lipoproteins and plasma lipoprotein free fraction (PLFF). Cardiolipin, phosphatidylglycerol (PG) and Phosphatidylserine (PS) were observed neither in the lipoprotein fractions nor in PLFF. All three approaches led to the same results regarding phospholipids occurrence in plasma lipoproteins and PLFF. A high abundancy of PE and SM was observed in VLDL and LDL fractions respectively. This study provides for the first time the knowledge about the phospholipid composition of all defined plasma lipoproteins.

Figures

Figure 1
Figure 1. Panel A shows the major human plasma lipoprotein fractions after one-step salt gradient ultracentrifugation.
Panel B shows Cholesterol profiles of major plasma lipoprotein fractions separated by High Performance Gel-Permeation Chromatography (HPGC). Lipoprotein fractions obtained by one-step ultracentrifugation were subjected to size-exclusion chromatography as described in Material and Methods. Panel (A) CM fraction, Panel (B) VLDL fraction, panel (C) LDL fraction, panel (D) HDL fraction, panel E: Plasma lipoproteins free fraction and panel F: standard cholesterol.
Figure 2
Figure 2. Protein profile of IDL fraction.
The ultra collected IDL fraction were several times exposed to HPGC to purify the IDL, therefore we used protein profile (to re-use elution for HPGC exposure) instead of cholesterol profile as described in Material and Methods. However, the cholesterol profile of ultra collected IDL fraction (before purification with HPGC) revealed approximately the same elution peak.
Figure 3
Figure 3. Western blot analysis of purified plasma lipoproteins.
Plasma lipoprotein- associated proteins (20–100 μg) were separated by 6 % (w/v) SDS-PAGE under denatured conditions, blotted for 20 h and probed with monoclonal anti-apo B antibodies as described in Materials and Methods. The plasma lipoproteins used were CM (lane 2, 80 ug), VLDL (lane 3, 80 ug), IDL (lane 4, 80 ug), LDL (lane 5, 80 ug), Apo B protein (lane 6, 20 ug), and HDL (lane 7, 100 ug). The lane 1 is protein marker and lane 8 is plasma lipoprotein free fraction (PLFF, 100 ug). The molecular weight standards (Mw) are in kilo Daltons (kDa) and indicated on the left of the figure. The approximately 500 kDa band is indicated by arrow on the left of the figure.
Figure 4
Figure 4. High-performance liquid chromatography electrospray ionization tandem mass spectrometry analysis of plasma lipoproteins and plasma lipoprotein free fraction.
We did not observe any adducts ions like Na or K. positive scan [M + H]+. Characteristic part of the parent-scan for phosphatidylethanolamine (PE) in the corresponding retention time window. ‘36∶2' specifies the composition of the protonated molecular ion as a species with 36 C-atoms in total in the acyl-chains with 2 unsaturated bonds in total; other assignments are analogous. (A) CM fraction, (B) VLDL fraction, (C) IDL fraction, (D) LDL fraction, (E) HDL fraction and (F) PLFF fraction.
Figure 5
Figure 5. High-performance liquid chromatography electrospray ionization tandem mass spectrometry analysis of plasma lipoproteins and plasma lipoprotein free fraction.
We did not observe any adducts ions like Na or K. positive scan [M + H]+. Characteristic part of the parent-scan for phosphatidylinositol (PI) in the corresponding retention time window. ‘36∶2' specifies the composition of the protonated molecular ion as a species with 36 C-atoms in total in the acyl-chains with 2 unsaturated bonds in total; other assignments are analogous. (A) CM fraction, (B) VLDL fraction, (C) IDL fraction, (D) LDL fraction, (E) HDL fraction and (F) PLFF fraction.
Figure 6
Figure 6. High-performance liquid chromatography electrospray ionization tandem mass spectrometry analysis of plasma lipoproteins and plasma lipoprotein free fraction.
We did not observe any adducts ions like Na or K. positive scan [M + H]+. Characteristic part of the parent-scan for sphingomyeline (SM) in the corresponding retention time window. ‘34∶1' specifies the composition of the protonated molecular ion as a species with 34 C-atoms in total in the acylchain and alkyl chain of the sphingosine moiety with 1 unsaturated bond in total; other assignments are analogous. (A) CM fraction, (B) VLDL fraction, (C) IDL fraction, (D) LDL fraction, (E) HDL fraction and (F) PLFF fraction.
Figure 7
Figure 7. High-performance liquid chromatography electrospray ionization tandem mass spectrometry analysis of plasma lipoproteins and plasma lipoprotein free fraction.
We did not observe any adducts ions like Na or K. positive scan [M + H]+. Characteristic part of the parent-scan for phosphatidylcholine (PC) in the corresponding retention time window. ‘34∶2' specifies the composition of the protonated molecular ion as a species with 34 C-atoms in total in the acyl-chains with 2 unsaturated bonds in total; other assignments are analogous. (A) CM fraction, (B) VLDL fraction, (C) IDL fraction, (D) LDL fraction, (E) HDL fraction and (F) PLFF fraction.
Figure 8
Figure 8. High-performance liquid chromatography electrospray ionization tandem mass spectrometry analysis of plasma lipoproteins and plasma lipoprotein free fraction.
We did not observe any adducts ions like Na or K. positive scan [M + H]+. Characteristic part of the parent-scan for lyso-phosphatidylcholine (LPC) in the corresponding retention time window. ‘18∶0' specifies the composition of the protonated molecular ion as a species with 18 C-atoms in the acyl-chain with 0 unsaturated bonds in total; other assignments are analogous. (A) CM fraction, (B) VLDL fraction, (C) IDL fraction, (D) LDL fraction, (E) HDL fraction and (F) PLFF fraction.
Figure 9
Figure 9. Separation of phospholipids from plasma lipoprotein fractions and plasma lipoprotein free fraction by HPTLC.
Plasma lipoprotein phospholipid composition and PLFF fraction were extracted and applied to one-dimensional, horizontal HPTLC as described under Materials and Methods. Representative separations are shown of a mixture of purified (ST = standard) phospholipids (lane 1 (10 μl), 2 and 12 (20 μ)). The following concentration of phospholipids (PL) were used in standard mixture and expressed in ng/μl (Lyso-PC 51, SM 101.8, PC 100, PI 50, PE 49.7, Lyso-PS 67.2, Lyso-PE 53 and PS 50.2). The following volumes of lipoprotein phospholipids extracts were utilized in HPTLC (Chylomicrons (lane 3, 3 μl), VLDL (lane 4, 4 μl), IDL (lane 5, 5 μl), LDL (lane 6, 2 μl), HDL2 (lane 7, 2 μl), PLFF (lane 8, 5 μl), Lp(a) (lane 9, 2 μl), HDL3 (lane 10, 4 μl), and VHDL (lane 11, 5 μl). The different phospholipids composition of standard mixture is indicated on the left and lane number on the top of figure.

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