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1.
Figure 3

Figure 3. Immunodetection of vitronectin (A), α-2-HS glycoprotein (B) and α-1–intertrypsin inhibitor (C) in HDL3. . From: Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL.

HDL3 was isolated from the plasma of 3 subjects by ultracentrifugation. HDL-associated proteins were separated under denaturing conditions by gel electrophoresis, transferred to a PVDF membrane, and subjected to immunoblot analysis. P, plasma.

Tomas Vaisar, et al. J Clin Invest. 2007 March 1;117(3):746-756.
2.
Figure 6

Figure 6. Reconstructed ion chromatograms of peptides derived from apoC-IV and apoA-I in HDL3. . From: Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL.

Tryptic digests of HDL3 isolated from CAD subjects and control subjects were subjected to LC-ESI-MS/MS analysis as described in the legend to Figure 1. Reconstructed ion chromatograms of peptides (arrows) unique to apoC-IV and apoA-I were extracted from the full-scan mass survey spectra of each analysis. cps, counts per second.

Tomas Vaisar, et al. J Clin Invest. 2007 March 1;117(3):746-756.
3.
Figure 5

Figure 5. Relative abundance of proteins isolated from HDL3 of control and CAD subjects. . From: Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL.

HDL3 was isolated from plasma of 6 control subjects and 7 subjects with established CAD. The relative abundance of proteins in the CAD subjects versus the control subjects was assessed by the peptide index, as described in Methods. *P < 0.05 by Student’s t test (peptide number) or Fisher’s exact test (subject number).

Tomas Vaisar, et al. J Clin Invest. 2007 March 1;117(3):746-756.
4.
Figure 8

Figure 8. Quantification of HDL-cholesterol, apoA-I, apoA-II, and apoE in HDL3 isolated from control and CAD subjects matched for plasma levels of triglycerides. . From: Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL.

Plasma was obtained from 32 apparently healthy individuals and 32 individuals with established coronary artery disease (group 3; Table 1). HDL3 was isolated from plasma by sequential ultracentrifugation. apoA-I, apoA-II, and apoE were quantified by ELISA. Results are normalized to the protein content of HDL3. *P = 0.02 by a 2-tailed Student’s t test.

Tomas Vaisar, et al. J Clin Invest. 2007 March 1;117(3):746-756.
5.
Figure 7

Figure 7. Relationship between unique peptides and protein concentration in HDL and HDL3. . From: Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL.

The total number of unique peptides detected by MS in HDL and HDL3 is as shown in Table 2. The protein concentrations of apoA-I (98 mg/dl), apoA-II (42 mg/dl), apoC-II (4.6 mg/dl), apoB (1.7 mg/dl), and transferrin (0.02 mg/dl) in HDL3 and HDL isolated by ultracentrifugation are those reported by McPherson et al. (where HDL equals the sum of HDL2 and HDL3; ref. 41). The concentration of CETP in HDL3 (0.1 mg/dl) was estimated as 50% that of plasma CETP (40). Linear regression analysis revealed a strong correlation (r2 = 0.98 and 0.88) between protein concentration and unique peptides in HDL and HDL3, respectively.

Tomas Vaisar, et al. J Clin Invest. 2007 March 1;117(3):746-756.
6.
Figure 4

Figure 4. Peptide number and number of subjects with detectable peptides in HDL3 isolated from control subjects and CAD subjects. . From: Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL.

HDL3 was isolated from the plasma of 6 control subjects and 7 subjects with established CAD (group 2; Table 1). HDL-associated proteins were subjected to LC-ESI-MS/MS analysis. All protein identifications required detection of at least 2 unique peptides from each protein from at least 2 individuals (Table 1). The P value was assessed by Student’s t test (peptide number) or Fisher’s exact test (subject number). *P < 0.05.

Tomas Vaisar, et al. J Clin Invest. 2007 March 1;117(3):746-756.
7.
Figure 2

Figure 2. Mass spectrometric and immunological detection of C3 in HDL3. . From: Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL.

HDL3 or apoA-I complexes were isolated from plasma of control subjects. In parallel studies, plasma was passed over columns containing albumin or nonspecific immunoglobulin covalently cross-linked to beads. Isolated proteins were separated under denaturing conditions by gel electrophoresis, transferred to nitrocellulose, and analyzed through immunoblotting. (A and B) MS/MS spectra of 2 peptides unique to C3 in HDL3. (C) Sequence of the C3dg domain of human complement C3. The 3 tryptic digest peptides detected selectively in HDL3 of CAD subjects are underlined. (D) Detection of proteins reactive with antibody to C3. Lane 1: plasma. Lanes 2 and 3: eluate from control columns (albumin and IgG, respectively). Lane 4: apoA-I complexes. Lanes 5–7: 3 independent preparations of HDL3 isolated from pooled human plasma.

Tomas Vaisar, et al. J Clin Invest. 2007 March 1;117(3):746-756.
8.
Figure 1

Figure 1. Global view of biological processes and molecular functions of HDL proteins.. From: Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL.

Proteins in total HDL and HDL3 were identified using LC-ESI-MS/MS (Table 2). Proteins detected in HDL were associated with biological functions using GO process annotations. This approach demonstrated significant overrepresentation of proteins involved in several categories, including lipid metabolism (P = 2 × 10–27), the acute-phase response (P = 1 × 10–18), protease inhibitor activity (P = 2 × 10–6), and complement regulation (P = 5 × 10–5). apoH, β-2-glycoprotein I; AGT, angiotensinogen; AHSG, α-2-HS-glycoprotein; AMP, bikunin; FGA, fibrinogen; HRP, haptoglobin-related protein; HPX, hemopexin; ITIH4, inter-α-trypsin inhibitor heavy chain H4; KNG1, kininogen-1; LCAT, lecithin-cholesterol acyltransferase; ORM2, α-1-acid glycoprotein 2; PLTP, phospholipid transfer protein; RBP4, retinol binding protein; SERA1, α-1-antitrypsin; SERF1, serpin peptidase inhibitor (clade F, member 1); SERF2, α-2-antiplasmin; TF, transferrin; TTR, transthyretin; VTN, vitronectin.

Tomas Vaisar, et al. J Clin Invest. 2007 March 1;117(3):746-756.

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