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. 2023 Jun 29;22(1):86.
doi: 10.1186/s12944-023-01829-9.

Proteomic and functional analysis of HDL subclasses in humans and rats: a proof-of-concept study

Canxia Huang #  1   2 , Jie Zhang #  1   3 , Jingjing Huang  1   3 , Hongwei Li  1   3 , Kexin Wen  1   3 , Jinlan Bao  1   4 , Xiaoying Wu  1   3 , Runlu Sun  1   3 , Ayiguli Abudukeremu  1   3 , Yue Wang  1   3 , Zhijian He  1   3 , Qiaofei Chen  1   3 , Xinyi Huang  1   3 , Hong Wang  5 , Yuling Zhang  6   7
Affiliations

Proteomic and functional analysis of HDL subclasses in humans and rats: a proof-of-concept study

Canxia Huang et al. Lipids Health Dis. .

Abstract

Background: The previous study investigated whether the functions of small, medium, and large high density lipoprotein (S/M/L-HDL) are correlated with protein changes in mice. Herein, the proteomic and functional analyses of high density lipoprotein (HDL) subclasses were performed in humans and rats.

Methods: After purifying S/M/L-HDL subclasses from healthy humans (n = 6) and rats (n = 3) using fast protein liquid chromatography (FPLC) with calcium silica hydrate (CSH) resin, the proteomic analysis by mass spectrometry was conducted, as well as the capacities of cholesterol efflux and antioxidation was measured.

Results: Of the 120 and 106 HDL proteins identified, 85 and 68 proteins were significantly changed in concentration among the S/M/L-HDL subclasses in humans and rats, respectively. Interestingly, it was found that the relatively abundant proteins in the small HDL (S-HDL) and large HDL (L-HDL) subclasses did not overlap, both in humans and in rats. Next, by searching for the biological functions of the relatively abundant proteins in the HDL subclasses via Gene Ontology, it was displayed that the relatively abundant proteins involved in lipid metabolism and antioxidation were enriched more in the medium HDL (M-HDL) subclass than in the S/L-HDL subclasses in humans, whereas in rats, the relatively abundant proteins associated with lipid metabolism and anti-oxidation were enriched in M/L-HDL and S/M-HDL, respectively. Finally, it was confirmed that M-HDL and L-HDL had the highest cholesterol efflux capacity among the three HDL subclasses in humans and rats, respectively; moreover, M-HDL exhibited higher antioxidative capacity than S-HDL in both humans and rats.

Conclusions: The S-HDL and L-HDL subclasses are likely to have different proteomic components during HDL maturation, and results from the proteomics-based comparison of the HDL subclasses may explain the associated differences in function.

Keywords: Antioxidation; Cholesterol efflux; Fast protein liquid chromatography; HDL subclass; High density lipoprotein; Proteomics.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
The strategies for identifying replicated HDL proteins. a The strategy for identifying replicated HDL proteins in rat samples. b The strategy for identifying replicated HDL proteins in human samples
Fig. 2
Fig. 2
Validation of three Superdex 200 columns plus CSH purification for HDL subclasses. a SDS‒PAGE of the HDL fractions in human and rat samples. FPLC-isolated HDL subclasses were analyzed by SDS‒PAGE after incubation with CSH, which removed most of the non-phospholipid binding proteins. The gels were stained with Coomassie blue. Human HDL was in fractions 7–24 and was divided evenly into the L-HDL subclass (fractions 7–12), M-HDL subclass (fractions 13–18), and S-HDL subclass (fractions 19–24). The rat HDL was in fractions 5–22 and was divided evenly into L-HDL subclass (fractions 5–10), M-HDL subclass (fractions 11–16), and S-HDL subclass (fractions 17–22). b Protein contents of the HDL fractions in humans and rats. The FPLC-isolated HDL fractions were all obtained and their protein contents were measured by absorbing at OD280
Fig. 3
Fig. 3
The identified proteins of the S/M/L-HDL subclasses in human and rat samples. a Heatmap of the ratios of the sum of peptide count for each protein in the S/M/L-HDL subclasses from human and rat samples. A equal volume of HDL subclasses were applied to the CSH resin, trypsinized, and then identified via LC‒MS/MS. The ratios of the sum of peptide count for each protein in the three HDL subclasses were calculated according to the unique peptide count of MS data, and a value of 1.0 was assigned to the subclass containing the highest peptide count for that particular protein and the peptide count of the other subclasses were scaled accordingly. the highest ratio (1.0) are colored red and gradually changed to yellow for the lower values, the lowest ratio (0.0) are colored green. The distinct proteins in the HDL subclass are shown in red font. b The distribution and overlaps of the proteins with the peptide count ratio of 1.0 among the HDL subclasses. The venn diagrams of the proteins with the peptide count ratio of 1.0 in each S/M/L-HDL subclass were drawn
Fig. 4
Fig. 4
The significantly changed proteins and dynamic protein distribution in the S/M/L-HDL subclasses from humans. a The significantly changed proteins in concentration between the HDL subclasses. The comparison of protein contents were analyzed by R, and the cutoff values of log2FC and -log10(P value) were 1.5 and 3.0, respectively. Log2 FC represents the logarithm value of fold change of the proteins that significantly changed in concentration between the S/M/L-HDL subclasses. b The dynamic distribution of the significantly changed proteins during HDL maturation in human samples. The dynamic distribution of the S-HDL to M-HDL subclasses and the M-HDL to L-HDL subclasses are indicated by the solid arrow, and the dynamic distribution of the S-HDL to L-HDL subclasses is indicated by the dotted arrow
Fig. 5
Fig. 5
The significantly changed proteins and dynamic protein distribution in the S/M/L-HDL subclass from rats. a The significantly changed proteins in concentration between the HDL subclasses. The comparisons of protein contents were analyzed by R, and the cutoff values of log2FC and -log10(P value) were 1.5 and 3.0, respectively. Log2 FC represents the logarithm value of fold change for the proteins that significantly changed in concentration between the S/M/L-HDL subclasses. b The dynamic distribution of the significantly changed proteins in concentration during HDL maturation in rats samples. The dynamic distribution of the S-HDL to M-HDL subclasses and the M-HDL to L-HDL subclasses are indicated by the solid arrow, and the dynamic distribution of the S-HDL to L-HDL subclasses is indicated by the dotted arrow
Fig. 6
Fig. 6
Overlapping and distinct proteins of the relatively abundant proteins in each S/M/L-HDL subclass from human and rats. a Venn diagram of the significantly changed proteins in concentration in the HDL subclasses of human and rat samples. b Venn diagram of the relatively abundant proteins in each human S/M/L-HDL subclass. c Venn diagram of the relatively abundant proteins in each rat S/M/L-HDL subclass. d Venn diagram of the relatively abundant proteins in the L-HDL subclass in human and rat samples. e Venn diagram of the relatively abundant proteins in the M-HDL subclass in human and rat samples. f Venn diagram of the relatively abundant proteins in the S-HDL subclass in human and rat samples
Fig. 7
Fig. 7
Functional annotation of the relatively abundant proteins in each HDL subclass in human and rats. The relatively abundant proteins in the S/M/L-HDL subclasses, as shown in Table 1, were searched in Gene Ontology (GO) annotations website (https://www.ebi.ac.uk/QuickGO/annotations), and the GO-molecular function and GO-biological process were grouped under ten umbrella terms. The relatively abundant proteins of the HDL subclasses are represented by different colored bars, and if none of the relatively abundant proteins in the HDL subclass grouped in GO terms, the proteins were represented by blank bars. The length of the bar represents the enriched log2FC value of the protein in each HDL subclass, and the critical maximum of the log2FC value (value 15) is marked as ∞
Fig. 8
Fig. 8
Cholesterol efflux and antioxidative capacities of each HDL subclass in human and rat samples. a Cholesterol efflux capacity of the S/M/L-HDL subclasses in human and rat samples. HDL subclasses were separated by FPLC and prepared for cholesterol efflux examination. J774A.1 cells were preloaded with 3H-cholesterol (1 Ci/ml) for 24 h. 3H-cholesterol efflux was detected after incubation with a equal volume of HDL subclass for 4 h. b Antioxidative capacity of the S/M/L-HDL subclasses in human and rat samples. HDL antioxidative ability was reflected as the antioxidative index, which was calculated as the endpoint fluorescence of DHR incubated with ox-LDL corrected for the optical density and divided by the optical density of DHR coincubated with ox-LDL and a equal volume of HDL subclass. *, P < 0.05
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