Fig. 5: Quantitative phosphoproteomics reveals kinase activities across gastruloid development.
From: The proteomic landscape and temporal dynamics of human and mouse gastruloid development
a, The temporal dynamics of phosphorylated peptides across human gastruloid development. Rows indicate phosphosites, and columns sample type. The colour scale indicates the scaled TMTpro reporter ion abundance of individual phosphopeptides. b, Ridgeplots depicting the characteristic phosphorylation states within a given protein. c, Venn diagrams depicting the detection of phosphorylated proteins that are targets of pluripotency factors SOX2, POU5F1 and NANOG. The gene sets were curated from ref. 40. d, Phosphosites associated with downstream targets of pluripotency factors. The y axis indicates the log2(abundance ratio) of early (yellow) or late (red) gastruloids to primed RUES2-GLR ESCs. Mean abundance ratios are indicated with dots, and error bars represent the s.d. calculated from three biological replicates. e, Bar plots of scaled H2AX pS139/pS140 abundance changes across human ESCs and gastruloid developmental stages. Mean abundance ratios are indicated with dots, and error bars represent the s.d. calculated from three biological replicates. f, Validation of the differential phosphorylation state of pS139/pS140 (red) in primed H9 (left) and RUES2-GLR (right) ESCs. The blue channel indicates DAPI. Scale bars, 25 µm. g, Heatmap depicting the z-scores of kinase–substrate enrichment analysis. h, Representative examples of temporal phosphosite dynamics in comparison to their respective proteins and cognate kinases. The colour scale indicates the abundance z-score. ECT2 T359 was correlated with PRKCI, and ZFP36L1 S92 was strongly correlated with both MAPKAPK2 and AKT1. i, Network of kinases (circles) connecting to their substrates (rectangles). Pairs are annotated from PhosphositePlus. Edge colours indicate correlated (blue) or anticorrelated (red) relationships (absolute rPearson ≥ 0.5) between kinase and substrate phosphosite nodes. j, Representative images of 120-h gastruloids cultured with DMSO (left) and the MAPKAPK2 inhibitor MK2in1 (right). Fluorescent images depict SOX2-mCitrine expression. BF, brightfield. Scale bars, 200 µm. k, Fraction of multi-axis gastruloids when treated with DMSO (control) and MK2in1 (MAPKAPK2 inhibitor). Fractions were calculated from 16 independent gastruloid observations for each condition. l,m, Boxplots depicting the differences in gastruloid area (l) and fraction of SOX2+ cells (m) when treated with DMSO (n = 9) and 10 μM MK2in1 (n = 15). Boxplots show the median (centre line), 25th–75th percentiles (box), 1.5× the interquartile range (line; end points signify maxima and minima). Significance was determined using a two-sided standard t-test.
