Fig. 2: Identification of fat body (FB)-derived proteins in legs/muscle organ.
From: Proteomics of protein trafficking by in vivo tissue-specific labeling
a Promiscuous biotin ligase BirA*R118G or our newly engineered high-activity BirA*G3 biotinylates all proteins in a subcellular compartment of one organ and the biotinylated proteins traffic to other organs or body parts. b Biotinylated proteins are enriched and identified by mass spectrometry. c–i, FB was labeled using LPP-Gal4>BirA*G3-ER (endoplasmic reticulum), and biotinylated proteins from legs/muscle organs were analyzed by tandem mass-tag (TMT) mass spectrometry (MS). Wild-type(wt) legs were used as controls. See Supplementary Fig. 2bb for the parts of legs used. Flies were maintained with 50 µM biotin in food during adulthood. c, Leg log2(BirA*G3-ER/wt) TMT ratios in two replicates. Each point is n = 2 comparisons, mean log2TMT ratio. Enrichment score (E-S): number of comparisons (from 4) in which TMT-ratio > threshold (score 4 [dark red] is for most confident hits and 0 [black] is background). d As the E-S increases, the fraction of proteins with putative signal peptides (see “Methods” section) increases. E-S = 0 vs E-S ≥ 1: ****p = 7.00 · 10−57; E-S = 0 vs E-S ≥ 2: ****p = 1.95 · 10−75; E-S = 0 vs E-S ≥ 3: ****p = 2.17 · 10−82. e, f As the E-S increases, fraction of proteins enriched for adult-FB mRNA microarray15 expression increases (e), and the fraction of proteins enriched for larval carcass (FB-free muscle organ data set) FB mRNA microarray15 decreases (f). E-S = 0 vs E-S ≥ 1: ****p = 2.78 · 10−12; E-S = 0 vs E-S ≥ 2: ****p = 3.67 · 10−17; E-S = 0 vs E-S ≥ 3: ****p = 4.53 · 10−18. g, h Hits are enriched for mammalian adipocyte (g) but not myocyte (h) secretome orthologs (see “Methods” section). ****p = 0.00007, ***p = 0.0004, **p = 0.0090. i Model of identified proteins known to traffic from FB to imaginal discs or muscle organ and known and additional FB-secreted proteins (see “Methods” section). In d–h a chi square test was used. N.S. means not significant. In d, g, h the test was two-sided, while in e, f the contingency table had three outcomes (up, none, or down), and one- or two-sided nature of the test was not applicable. See also: Supplementary Figs. 4–8, Supplementary Tables 3–7, Supplementary Data 1–3. Source data are provided as a Source Data file.
