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. 2023 Jul 13;12:RP86972. doi: 10.7554/eLife.86972

Figure 5. Presence of glutamine in synaptic vesicles (SVs) from the mouse brain.

(A) A schematic diagram illustrating the procedure to immunoisolate SVs with the anti-Syp antibody for liquid chromatography coupled to mass spectrometry (LC-MS) analysis of the contents in the SVs. (B) Representative MS results showing Gln signals from SVs immunoisolated by the anti-Syp antibody (anti-Syp) vs. the control sample immunoisolated with IgG. (C) Volcano plot of chemical contents in the SVs isolated by anti-Syp vs. IgG. The y axis shows p-values in log10 and the x axis shows the log2 of the ratio of the level of a molecule immunoisolated by anti-Syp vs. IgG. Classical neurotransmitters Glu, GABA, and ACh, as well as previously reported substrates of SLC6A17 are listed. (D) Ratios of the level of a chemical immunoisolated by anti-Syp vs. IgG (transformed into log2). (E–I) Chemicals were quantified to mole per μg antibody (n = 12 for each group from four different animals with three replicates each): Glu (E, p<0.0001 for anti-Syp vs. IgG); GABA (F, p<0.0001 for anti-Syp vs. IgG); ACh (G, p<0.0001 for anti-Syp vs. IgG); Gln (H, p<0.0001 for anti-Syp vs. IgG); Ser (I, p=0.7553 for anti-Syp vs. IgG).

Figure 5—source data 1. Data points for Figure 5C–I.

Figure 5.

Figure 5—figure supplement 1. Liquid chromatography coupled to mass spectrometry (LC-MS) analysis of SVs immunoisolated by the anti-Syp antibody.

Figure 5—figure supplement 1.

(A) Analysis of SVs isolated by the anti-Syp antibody. 16 markers for organelles were analyzed to confirm the SV specificity, with no detection of markers for lysosome, Golgi, mitochondria, ER, endosome and postsynaptic cytoplasmic membrane. IgG was used as a control. (B–G) Neurotransmitter contents in SVs isolated by the anti-Syp antibody. Representative LC-MS signals of anti-Syp and IgG group are shown, each graph was normalized: Glu (B), GABA (D), and ACh (F) highly enriched; 5-HT (C), histamine (E), and dopamine (G) moderately enriched.
Figure 5—figure supplement 1—source data 1. Original files of the full raw unedited blots for Figure 5—figure supplement 1A.
Figure 5—figure supplement 1—source data 2. Uncropped blots with the relevant bands labeled for Figure 5—figure supplement 1A.