1. Cell-surface tethered promiscuous biotinylators enable comparative small-scale surface proteomic analysis of human extracellular vesicles and cells
- Author
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Kirkemo, Lisa L, Elledge, Susanna K, Yang, Jiuling, Byrnes, James R, Glasgow, Jeff E, Blelloch, Robert, and Wells, James A
- Subjects
Biochemistry and Cell Biology ,Biological Sciences ,Cancer ,Bioengineering ,Extracellular Vesicles ,Horseradish Peroxidase ,Humans ,Male ,Proteome ,Proteomics ,Wheat Germ Agglutinins ,extracellular vesicle ,HRP ,APEX2 ,proteomics ,surfaceomics ,cell surface ,Human ,biochemistry ,cancer biology ,chemical biology ,human ,Biological sciences ,Biomedical and clinical sciences ,Health sciences - Abstract
Characterization of cell surface proteome differences between cancer and healthy cells is a valuable approach for the identification of novel diagnostic and therapeutic targets. However, selective sampling of surface proteins for proteomics requires large samples (>10e6 cells) and long labeling times. These limitations preclude analysis of material-limited biological samples or the capture of rapid surface proteomic changes. Here, we present two labeling approaches to tether exogenous peroxidases (APEX2 and HRP) directly to cells, enabling rapid, small-scale cell surface biotinylation without the need to engineer cells. We used a novel lipidated DNA-tethered APEX2 (DNA-APEX2), which upon addition to cells promoted cell agnostic membrane-proximal labeling. Alternatively, we employed horseradish peroxidase (HRP) fused to the glycan-binding domain of wheat germ agglutinin (WGA-HRP). This approach yielded a rapid and commercially inexpensive means to directly label cells containing common N-Acetylglucosamine (GlcNAc) and sialic acid glycans on their surface. The facile WGA-HRP method permitted high surface coverage of cellular samples and enabled the first comparative surface proteome characterization of cells and cell-derived small extracellular vesicles (EVs), leading to the robust quantification of 953 cell and EV surface annotated proteins. We identified a newly recognized subset of EV-enriched markers, as well as proteins that are uniquely upregulated on Myc oncogene-transformed prostate cancer EVs. These two cell-tethered enzyme surface biotinylation approaches are highly advantageous for rapidly and directly labeling surface proteins across a range of material-limited sample types.
- Published
- 2022