Fig. 2: Differences between iSPN and dSPN postsynaptic compartment proteomes are predominantly at the isoform level.

a Experimental design to compare iSPN and dSPN postsynaptic compartment proteomes. n = 5 biological replicates (BRs) for each genotype. b Biological replicates cluster by genotype in (t-distributed stochastic neighbor embedding) tSNE plot based on protein analysis. c Volcano plot depicting comparison of iSPN and dSPN postsynaptic compartment proteomes based on protein analysis. Pie chart showing only ~5% proteins in iSPN postsynaptic compartment proteomes are differentially expressed in dSPNs. For proteins with multiple UniProt accessions, we only considered the accession with highest total TMT reporter ion intensity. See Sheet1 in Table S2 for exact p value and the iSPN/dSPN ratio for each postsynaptic protein in the protein analysis. One-tailed Student’s t test. d Isoform analysis revealed that the postsynaptic proteins were divergently expressed in iSPNs and dSPNs at isoform level. For protein analysis, p values were calculated by One-tailed Student’s t test. For isoform analysis, p-values were calculated by two-way ANOVA. (PR > F): Genotype * peptide values were used. See Sheet2 in Table S2 for exact p vlaue: C(Genotype), p-vlaue: C(Peptide) and p-vlaue: C(Genotype):C(Peptide) for each postsynaptic protein in the isoform analysis. e Gene functional classification analysis of postsynaptic proteins isoform-divergently expressed in iSPNs and dSPNs. For all listed terms, count > 30, adj. p value < 0.05. f Top, p values acquired by two-way ANOVA analysis of Shank3 peptides. Bottom, quantile-quantile (Q-Q) plots showing that the variances of Shank3 peptides in iSPNs and dSPNs are heteroscedastic. g Experimental design of GeLC-MS² based Shank3 protein isoform profiling. h Representative Coomassie blue (CBB) staining gels of NeutrAvidin affinity purified material from iSPN and dSPN samples. i Heatmap showing the distribution pattern of all identified Shank3 peptides in gel slices. See Table S3 for the exact sequence and mapped exon(s) of each identified Shank3 peptide in GeLC-MS² experiment. j Profiling of Shank3 protein isoforms in iSPNs and dSPNs postsynaptic compartments. Left, in gel slice I, the Shank3 protein isoform(s) expressed in iSPNs were closely-clustered with Shank3A. In contrast, the isoform(s) expressed in dSPNs were separated from others, suggesting there is unreported long Shank3 isoform(s) co-expressed with Shank3A in dSPNs. Right, in gel slice II, the isoforms expressed in dSPNs and iSPNs were loosely clustered with Shank3E.