Figure 1. Direct labeling of promiscuous biotinylators to the cell membrane for rapid cell surface proteome characterization of small-scale biological samples.

(A) Outline of enzymatic reaction mechanism. APEX2 and HRP both require biotin tyramide and hydrogen peroxide to produce the biotin-radical intermediate. (B) Tethering either enzyme is completed through differing mechanisms: (i) APEX2 is tethered through bio-conjugation of a single-strand of DNA, which is complementary to an exogenously added sequence of lipidated-DNA attached to the membrane, (ii) Commercially available wheat germ agglutinin (WGA)-HRP associates with native GlcNAc and sialic acid glycan moieties on cell surface proteins. (C) Microscopy images depicting the localization of DNA-APEX2 to the cell surface of KP-4 cells after the introduction of the lipidated-DNA complementary strands. (D) Microscopy images depicting the localization of WGA-HRP to the membrane of KP-4 cells. All microscopy images are representative of two biological replicates.

Figure 1—figure supplement 1. Expression, purification, and validation of APEX2 enzyme.

His-tagged APEX2 was expressed in BL21(DE3)pLysS cells and purified by a nickel column. Ten milligrams of the purified enzyme were run out on a 4%–12% Bis-Tris gel to confirm purity.

Figure 1—figure supplement 2. Labeling and efficacy of APEX2 with DNA.

(A) APEX2 was first conjugated with DBCO-Maleimide (DBCO-Mal) reagent at 40 equivalents for 5 hr (80% conversion to the singly labeled product). Following desalting, three equivalences of Azide-DNA were added to the conjugate and purified by a Nickel column. Both reactions were monitored by LC-MS as shown. (B) 500,000 Expi293 cells were labeled with 0.5 µM purified APEX2 and DBCO-labeled APEX2 for 2 min. Extent of biotinylation of target cells was quantified by flow cytometry staining with streptavidin-647. (C) The DNA-APEX2 conjugate was shown to be tethered in the presence of the lipidated DNA (purple) and not in the absence (green), as detected by an Anti-His 680 antibody. Unlabeled APEX2 (blue) additionally did not result in a signal shift. Flow cytometry images are representative of one biological replicate.

Figure 1—figure supplement 3. WGA-HRP preincubation time on cells has no effect on labeling efficiency.

WGA-HRP was incubated on Expi293 cells for 0–30 min to determine optimal incubation time on ice before labeling. All tested times resulted in similar cell surface biotinylation efficiencies and signified that no incubation time was needed. Flow cytometry images are representative of one biological replicate.