G2C::Proteomics

Figure 1. Validation of fractionation method and quantitative comparison of mouse and human cortical PSDs.

a. Immunoblots of known postsynaptic density components. Mouse synaptosomes (S) and PSD (P) fractions are analysed for each of the three replicates. All proteins show enrichment in the PSD compared to synaptosomes. The presynaptic marker synaptophysin (Syn) is only detected in the synaptosomal fraction.

Only genes identified in triplicate in both species were considered for the quantitative analysis. Correlations are calculated based on linear models. Coefficients of correlations are represented by r.

b. Intra-species protein abundance correlation, human PSD first replica against second

c. Intra-species protein abundance correlation, mouse PSD first replica against second.

d. Plot of average values of PSD proteins abundance from human and mouse cortex.

e. Protein abundance correlation of PSD components found in the NMDA receptor complex [3];

f. Protein abundance correlation of PSD components found in the PSD-95 complex [26].

g.Protein abundance correlation of PSD components found in the mGluR5 complex [27].

h. Clustering of PSD protein abundance values for human and mouse replicates.

Figure 2. Analysis of evolutionary conservation of PSD proteins in mouse and humans.

a.Median dN/dS of proteins identified in the human PSD, mouse PSD and mouse consensus set (cPSD) compared with the median genomic value.

b.Comparison of dN/dS values in proteins exclusive or enriched in one species with all PSD proteins.

Figure 3. Inter-species differences in postsynaptic density proteins.

a.Venn diagrams showing the total amount of proteins in mPSD and hPSD as well as the overlap between them relative to the hPSD.

b.Venn diagrams showing the proteins identified in triplicate for the mPSD and hPSD, the overlap relative to hPSD between the two sets is also shown.

c.Immunoblot validation of proteins identified only in the hPSD by mass spectrometry. The following samples were analysed: H-M, mouse brain homogenate; H-H, human brain homogenate; and SX, each of the three synaptosomal fractions, PX, each of the three postsynaptic density fractions for both species.

d.The abundance fold difference (AFD) between mouse and human is shown for members of adaptors, channels and neurotransmitter receptors protein families. AFD is calculated as the mouse to human ratio of normalized and log₂ transformed average abundance values. For each protein family the number of proteins displayed is given in brackets.

Figure 4. Bioinformatic functional analysis of proteins enriched in the human PSD.

a.Ontology hierarchical tree leading to the Biological Process ‘Cellular Component Morphogenesis’. In red are the terms significantly enriched in proteins abundant in the human PSD.

b.Molecular interaction map of proteins from the human PSD that belong to the Biological Process ‘Cellular Component Morphogenesis’ (CCM) or are known to be involved in semaphorin signaling. Only CCM proteins enriched in the human PSD are considered. Some proteins related to semaphorin signaling are not enriched in either species, these are identified with a white name. Proteins highly interconnected are represented in clusters of different colors. Clustering was done using the Markov Clustering Algorithm (MCL) in BioLayout Express 3D [24]. Node edges are coloured according to a confidence score, from red (100% confidence) to blue (40% confidence).

Figure 5. Predicted functional protein groups with the mammalian cortical PSD.

The mammalian cortical PSD was defined by combining all proteins identified in mouse and human. This set of PSD proteins were then functionally classified using the Panther Classification descriptor ‘Protein Class’. Enrichment analysis was done comparing the number of PSD proteins per class with the genome. The Panther Protein Class ontology is hierarchically organized, only end-branch classes are displayed to remove redundancy from the graph (all data in Table S8). Protein classes that did not show difference with the genome after correction for multiple testing, are shown in blue. Classes overrepresented are shown in red and classes underrepresented are shown in green.

Table 1. Significantly enriched protein classes of species-specific PSD proteins.

Table 2. Abundance of canonical PSD components as defined by studies with rodent brains.

Mouse to human abundance difference of proteins known to be key components of the postsynaptic density, as derived from studies on mouse and rat brains. Abundance is expressed as the fold of mouse to human average abundance values.

Supplementary Table 1. Protein Identifications and Proteomic Data.

For each identified protein several identification (ID) numbers from biological databases are given. The number of total and uniquely identified peptides for each replicate is also provided. Proteins found with two or more peptides in all replicates are classified as members of the consensus mouse PSD. Human orthologues to mouse proteins are provided. Data regarding human PSD proteins is from: Bayes A et al. Nat Neurosci. 2011 Jan;14(1):19-21.