Figure 5. Analysis of the Presynaptic Transportome.

(A) Venn diagram comparing the ON transportome and presynaptic transportome. The presynaptic transportome contains many classical presynaptic proteins involved in presynaptic machinery for neurotransmitter release.

(B) Volcano plot comparing protein abundance in the ON transportome and the SC transportome. Significantly enriched proteins are in red. Data points plotted on the left or right y axes represent proteins that are only detected in one dataset. n = 3, p < 0.05, t test with Benjamini-Hochberg correction.

(C) Enrichment of presynaptic proteins, identified from the annotated presynaptic protein databases, in the ON, SC, and LGN transportomes normalized to the retina. The NeutrAvidin flow through (LGN fl) is not enriched in presynaptic proteins. Fisher’s exact test, **p < 0.001, †† < 0.001 (Z = −5.35, retina versus ON; Z = −9.9772, retina versus LGN transportome; Z = −11.1862, retina versus SC transportome; Z = −0.9256, p = 0.35238, retina versus total LGN; Z = −4.8628, ON transportome versus LGN transportome; and Z = −5.5885, ON transportome versus SC transportome).

(D) Western blot validation of biotinylated proteins from SC, with NeutrAvidin enrichment of MTOR, MUNC18, and ALIX, but not GFAP, in NHS-biotin treated animals versus control. SC tissue from 3 animals was pooled per sample. Experiments were done in triplicate.

(E) Confocal images of LGN from animals with intravitreal NHS-biotin show biotinylated proteins (green) in RGC presynaptic boutons, with either endogenous synaptotagmin 1 or ALIX (red). Images show colocalization of endogenous proteins with biotin label in presynaptic boutons. Scale bar: 50 μm.

(F) Venn diagram comparing the presynaptic transportome with those of public databases for presynaptic proteins. Proteins detected only in the presynaptic transportome include those that function in vesicle trafficking and those involved in proteostasis.

Proteomic data used to generate these figures are shown in Table S3.