Cell-surface tethered promiscuous biotinylators enable comparative small-scale surface proteomic analysis of human extracellular vesicles and cells
- University of California, San Francisco, United States
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 (EV), 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.
Data availability
All data has been deposited alongside the manuscript as supporting source data files. Raw western blot images are made available as a source file titled "Raw_WesternBlot". All data from mass spectrometry experiment is provided as source data within the manuscript. "Figure 3-source data 2" details the quantification results from the RWPE-1 +/- Myc cell comparison experiments. "Figure 4-source data 2" details the quantification results from the RWPE-1 +/- Myc EV comparison experiments. "Figure 4-source data 3" details the quantification results from the RWPE-1 +/- Myc whole EV experiments. "Figure 5-source data 2" details the quantification results from PEAKS and Perseus for the RWPE-1 +/- Myc EV and cell comparison experiments."PaTu8902_WGAvsAPEX2_DatabaseSearch" and "KP4_APEX_HRP_Comparison_DatabaseSearch" documents detail results from APEX2 and HRP method comparisons across two different PDAC cell lines."RWPE_Method_Comparison_DatabaseSearch" outlines the results from the NHS-biotin, biocytin hydrazide, and WGA-HRP comparison experiments performed on RWPE EV and Myc transduced cells.The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD028523.
Article and author information
Author details
Funding
National Cancer Institute (1F31CA247527)
- Lisa L Kirkemo
National Science Foundation (1650113)
- Susanna K Elledge
National Cancer Institute (U01CA244452)
- Jiuling Yang
- Robert Blelloch
National Institute of General Medical Sciences (R35GM122451)
- James A Wells
National Cancer Institute (R01CA248323)
- James A Wells
Chan Zuckerberg Biohub Investigator Program
- James A Wells
Harry and Dianna Hind Professorship
- James A Wells
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2022, Kirkemo et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Cell-surface tethered promiscuous biotinylators enable comparative small-scale surface proteomic analysis of human extracellular vesicles and cells
eLife 11:e73982.
https://doi.org/10.7554/eLife.73982
