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. 2022 Mar 8;11:e68148. doi: 10.7554/eLife.68148

Figure 1. Pulsed N-hydroxysuccinimidobiotin (NHS-biotin) labels and quantifies protein transport after injury.

(a) NHS-biotin (biotin group in blue) reacts with primary amines adding a defined mass shift to tagged proteins. (b) NHS-biotin (blue) was injected before injury with a repeat injection 21 hr later. Biotinylated proteins in retinal ganglion cells (RGCs) transport down intact RGC axons in the optic nerve. The retinas and optic nerves were collected for tissue processing after 3 hr. (c) No biotinylated proteins were detected by immunofluorescence distal to the crush site. Scale bar, 100 µm. (d) Pearson’s R correlation of proteomic replicates of control or optic nerve crush (ONC) samples (R = 0.805 and 0.914, respectively). (e) Ponceau S stain of protein from biotinylated retinas and optic nerves, with and without ONC, showing equivalent total protein. Molecular ladder labeled on left of blot. (f) Western blot of the same samples run in parallel, probing for biotin. ONC decreased biotinylated proteins to a greater extent in ON compared to retina. Molecular ladder labeled on left of blot.

Figure 1—source data 1. Proteomic quantification of transportome changes after ONC from Figure 1b.
Figure 1—source data 2. Raw western blots from Figure 1.

Figure 1.

Figure 1—figure supplement 1. Alternative protein labeling strategies.

Figure 1—figure supplement 1.

(a) NHS-azide was injected into the vitreous space. Twenty-four hours later, retinal and optic nerve tissue was collected for either biochemistry or immunofluorescence. Right panels: We found labeled cells across the surface of the retina after a click reaction with an Alexa-488 alkyne but did not detect label in the optic nerve. Retina scale bar, 20 µm, ON scale bar, 100 µm. Left panel: With a biotin alkyne click reaction, we were able to detect proteins in the optic nerve and show that they decreased after optic nerve crush, compared to a low background without a biotin alkyne click reaction. Bands in the control lane (right) are endogenous biotin-bound proteins. (b) Biotin-beta-alanine was injected intravitreously or provided to HEK cells in culture. Left panel: Biotinylated proteins of different masses were detectable by western blot from HEK cells. Right panels: After 24 hr, biotin-beta-alanine was detected in sections through the optic nerve, compared to optic nerve from animals with intravitreal PBS injection. Scale bar, 100 µm. Both techniques are valuable for biochemistry and histology, although protein labeling with N-hydroxysuccinimidobiotin (NHS-biotin) was superior for mass spectrometry.
Figure 1—figure supplement 1—source data 1. Raw western blots from Figure 1—figure supplement 1.