Journal: bioRxiv

Article Title: Mapping the dynamic cell surface interactome of high-density lipoprotein reveals Aminopeptidase N as modulator of its endothelial uptake

doi: 10.1101/2023.01.03.522574

Figure Lengend Snippet: Decoding the HDL-derived and cell-derived HDL synapse compositions across four independent experiments. A) Cumulative log2(FC) for indicated proteins in the four HDL-based LUX-MS experiments relative to corresponding controls. In each bar, the blue region indicates the ratio of light peptide intensities to heavy peptides (mean of two experiments). Proteins with a FC > 1.5 in all four experiments are highlighted in bold. Dots below the graph indicate whether the protein is part of the Surfy or the HDL proteome list. B) Correlation plot with log2(FC) of the enriched proteins in the four independent LUX-MS experiments with four different HDL isolates (HDL A - D). Green points indicate FC > 1.5 in all four experiments. (C) Number of proteins that reached a FC threshold > 1.5 in all four experiments. Dark grey indicates surface or HDL annotation for the protein and light grey indicates a cytosolic protein. Green bar indicates the 60 core candidates; all are on the Surfy or the HDL proteome list. D) Gene ontology analysis of the 60 core candidates compared to the endothelial cell surfaceome. Displayed are all significantly enriched terms (p value corrected with Bonferroni step down < 0.05). The size of the dots inversely corresponds to the mean gene ontology term level.

Article Snippet: HAECs (Cell Applications Inc (304-05a)) were cultured in Human Endothelial Cell Growth Medium, All-in-one ready-to-use (Cell Applications Inc, 211A-500) without antibiotics.

Techniques: Derivative Assay, Targeted Proteomics

Journal: bioRxiv

Article Title: Mapping the dynamic cell surface interactome of high-density lipoprotein reveals Aminopeptidase N as modulator of its endothelial uptake

doi: 10.1101/2023.01.03.522574

Figure Lengend Snippet: Interconnectivity of endothelial HDL synapse candidates and validation of AMPN as an important member of the interactome. A) Interaction network of the endothelial HDL synapse core candidates and proteins that are either up- or downregulated upon stable silencing of PECA1, TRBM, NECT2, SHPS1, or SCRB1. Edges represent either regulated proteins or STRING database confidence >0.7. B) Subset of network highlighting endothelial HDL core candidates that are regulated upon stable silencing of PECA1, TRBM, NECT2, SHPS1, or SCRB1 (i.e., FC > 1.5 or < −1.5 and adj. p value < 0.05). C) HDL association with HAECs in which either AMPN or SCRB1 were transiently silenced with siRNA. Data are from three independent experiments with quadruplicates or triplicates. Vertical lines indicate the minimum and maximum values, boxes are first and third quartiles, and horizontal lines are medians. Significance was determined with the Wilcoxon rank sum test (** p < 0.01; *** p < 0.001).

Article Snippet: HAECs (Cell Applications Inc (304-05a)) were cultured in Human Endothelial Cell Growth Medium, All-in-one ready-to-use (Cell Applications Inc, 211A-500) without antibiotics.

Techniques: Biomarker Discovery

Journal: bioRxiv

Article Title: Mapping the dynamic cell surface interactome of high-density lipoprotein reveals Aminopeptidase N as modulator of its endothelial uptake

doi: 10.1101/2023.01.03.522574

Figure Lengend Snippet: The hepatic HDL synapse and its comparison to the endothelial HDL synapse. A) Volcano plots of LUX-MS experiments using an anti-SCRB1 antibody (left) and HDL (right) as a ligand in LUX-MS experiments on HEPG2 cells. Proteins enriched in the anti-SCRB1 antibody-based LUX-MS experiment are in blue. Proteins enriched in the HDL-based LUX-MS experiment are in red. Proteins enriched in both experiments are in purple. B) Numbers of proteins detected as enriched in anti-SCRB1 antibody-based and HDL-based LUX-MS experiments and the number of overlapping proteins. C) Comparison of the HDL core synapse candidates on EA.hy926 cells (green, outer circle) and the enriched surface/ HDL-associated proteins on HEPG2 cells (orange, inner circle). Grey indicates proteins of the HEPG2 or EA.hy926 surfaceome that were not enriched in the LUX-MS experiment. Significance was determined using MSstats (FC > 1.5 and p value < 0.05). Note: There is no evidence that ECSCR, CD99, or GELS are N- glycosylated and therefore the presence on the cell surface cannot be excluded as auto-CSC targets N- glycosylated surface proteins.

Article Snippet: HAECs (Cell Applications Inc (304-05a)) were cultured in Human Endothelial Cell Growth Medium, All-in-one ready-to-use (Cell Applications Inc, 211A-500) without antibiotics.

Techniques: Comparison