Extended Data Fig. 4: Quality assessment of proteomic and phosphoproteomic analysis.
From: Quantitative phosphoproteomic analysis of the molecular substrates of sleep need
a, Representative TMT quantification spectrum for the pS551-containing phosphopeptide from the skipped Sik3 exon-13 among phosphoproteomic data of the Sleepy model (two independent experiments). b–e, Quality assessment of one proteomic dataset (EX4, SlpWTpa2) by two search pipelines. Global distribution of protein quantification using Proteome Discoverer (PD v.2.1; n = 8,273) (b) and JUMP (v.1.12.1; n = 8,473) (c). Boxes correspond to the 25th, 50th and 75th percentiles of the data, whiskers extend to 1.5-fold of the interquartile range. A similar number of accepted proteins (1% FDR) were identified by two pipelines (d). Pearson correlation between the two pipelines was calculated for each PSM from quantified proteins by both pipelines (e). The vast majority (99.88%) of PSMs (n = 73,454) have R2 values larger than 0.9 (red dashed line). f, A Venn diagram showing overlaps of quantified proteins between whole brain proteomes of Sleepy and sleep-deprived models. g, Volcano plots showing comparative analysis of Sleepy/wild-type, SD6/RS3 and SD6/S6 proteomes. Multiple unpaired t-test (P value) followed by FDR (Q value) analysis. x axis, log2(fold change) in abundance; y axis, −log(Q value) of abundance change. The numbers of total (n), increased (in: Q < 0.2, red) and decreased (de: Q < 0.2, blue) subjects are shown. Orange dotted lines indicate Q = 0.2. h, Pearson correlation between normalized and unnormalized phosphopeptides in Sleepy/wild-type, SD6/RS3, SD6/S6 groups. The numbers of phosphopeptides that can be normalized are shown. i, Immunoblots were performed with phosphorylation-site specific antibodies to verify hyper-phosphorylation of several proteins in two models. Three or two independent experiments for sleep-deprived or Sleepy models, respectively. j, Quantitative analysis of immunoblots in i, normalized with whole protein abundance, for Sleepy (n = 6) and sleep-deprived (n = 9) models. Mean ± s.d., two-way ANOVA with Fisher’s LSD test. *(black), P < 0.05; *(cyan), P < 0.01; *(red), P < 0.001; ns, P > 0.05.
