Correlation profiling of brain sub-cellular proteomes reveals co-assembly of synaptic proteins and subcellular distribution

Abstract

Protein correlation profiling might assist in defining co-assembled proteins and subcellular distribution. Here, we quantified the proteomes of five biochemically isolated mouse brain cellular sub-fractions, with emphasis on synaptic compartments, from three brain regions, hippocampus, cortex and cerebellum. We demonstrated the expected co-fractionation of canonical synaptic proteins belonging to the same functional groups. The enrichment profiles also suggested the presence of many novel pre- and post-synaptic proteins. Using super-resolution microscopy on primary neuronal culture we confirmed the postsynaptic localization of PLEKHA5 and ADGRA1. We further detected profound brain region specific differences in the extent of enrichment for some functionally associated proteins. This is exemplified by different AMPA receptor subunits and substantial differences in sub-fraction distribution of their potential interactors, which implicated the differences of AMPA receptor complex compositions. This resource aids the identification of proteins partners and subcellular distribution of synaptic proteins.

Figure 1

Biochemical isolation of cellular sub-fractions from three brain regions. Fractions labelled red were collected for proteomics analysis.

Figure 2

Hierarchical clustering of 3632 proteins shows specific enrichment in various subcellular fractions of the hippocampus. Protein mean abundances were scaled between zero and 100% of their maximum over all sub-fractions.

Figure 3

Protein abundance distribution over the cellular sub-fractions in hippocampus (HC), cortex (CT) and cerebellum (CB). Left panel is an immunoblot analysis showing the presence of presynaptic protein SYP, and postsynaptic proteins GRIN2A and DLG4 in each sub-fraction. Right panel shows protein abundance in each sub-fraction as observed with proteomics. Mass spectrometry protein abundance values were scaled between zero and 100% of their maximum over all sub-fractions in each panel and accordingly color-coded from light-blue to dark-blue.

Figure 4

Correlation analysis of abundance profiles of proteins over sub-fractions of the hippocampus with selected seed proteins. In each panel, three functionally related seed proteins were chosen typical for each of the specific synaptic processes in (A) and (B). Proteins shown have a Pearson correlation of at least 0.9 with at least two out of these three seed proteins. Protein abundances are scaled between zero and their maximum intensity over all selected sub-fractions. This leads to A) exocytosis, 411 proteins and B) postsynaptic density, 346 proteins.

Figure 5

Comparison of the 49 PSD-95 (DLG4)-affinity associated proteins as listed in Table 2 from Dosemeci et al.. (A) 31 proteins from this reference set are recovered by correlation analysis with hippocampal sub-fractions (as used for Fig. 4B). (B) 8 additional reference proteins are recovered using the Pearson correlation threshold from 0.9 to 0.5 (for 2 + reference proteins). (C) 10 remaining proteins from the reference set which were not recovered by correlation analysis. Summed iBAQ abundance of each protein is shown alongside each protein name. Protein abundances were scaled between zero and 100% of their maximum over all sub-fractions and color-coded from light-blue to dark-blue according to the heatmap.

Figure 6

The sub-cellular abundance of ionotropic glutamate receptors and their associated proteins in three brain regions. (A) AMPA receptor subunits GRIA1-4, and 6 known auxiliary subunits, compared between sub-fractions and brain regions. Each group in the bar graph reflects the abundance of a protein over sub-fractions. (B) NMDA receptor subunits GRIN1 and GRIN 2 A/B, and the interactors DLG4, DLGAP4, HOMER1 and SHANK1. Legend at the bottom reflects the order of sub-fractions and their respective gray scale coding. Protein iBAQ values are computationally approximated absolute abundances.

Figure 7

Super-resolution imaging microscopy validation of novel PSD-enriched proteins, PLEKHA5 and ADGRA1. (A,E) SIM imaging of primary cultured hippocampal neurons at DIV19 for PLEKHA5 and ADGRA1 (green) along with HOMER1 (red), the latter as a marker for the PSD. Lower panel shows zoom in of the marked area (scale bar 2 µm). (B,C) Line scan analysis on inset from panel a,e shows co-localization between the three proteins at postsynaptic sites. (D) Bar graph showing percentage (mean ± sem) of postsynaptic density HOMER1 puncta positive for PLEKHA5 (dark green, 48.61 ± 4.92, n = 10), ADGRA1 (light green 96.54 ± 1.02, n = 7).