Abstract
Detailed observations of transcriptional, translational and post-translational events in the human brain are essential to improving our understanding of its development, function and vulnerability to disease. Here, we exploited label-free quantitative tandem mass-spectrometry to create an in-depth proteomic survey of regions of the postnatal human brain, ranging in age from early infancy to adulthood. Integration of protein data with existing matched whole-transcriptome sequencing (RNA-seq) from the BrainSpan project revealed varied patterns of protein–RNA relationships, with generally increased magnitudes of protein abundance differences between brain regions compared to RNA. Many of the differences amplified in protein data were reflective of cytoarchitectural and functional variation between brain regions. Comparing structurally similar cortical regions revealed significant differences in the abundances of receptor-associated and resident plasma membrane proteins that were not readily observed in the RNA expression data.
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Acknowledgements
We thank S. Leslie and D. Li for discussions. Data were generated as part of the PsychENCODE Consortium, supported by U01MH103339, U01MH103365, U01MH103392, U01MH103340, U01MH103346, R01MH105472, R01MH094714, R01MH105898, R21MH102791, R21MH105881, R21MH103877 and P50MH106934 awarded to S. Akbarian (Icahn School of Medicine at Mount Sinai), G. Crawford (Duke University), S. Dracheva (Icahn School of Medicine at Mount Sinai), P. Farnham (University of Southern California), M.B.G. (Yale University), D. Geschwind (University of California, Los Angeles), T.M. Hyde (Lieber Institute for Brain Development), A. Jaffe (Lieber Institute for Brain Development), J.A. Knowles (University of Southern California), C. Liu (University of Illinois at Chicago), D. Pinto (Icahn School of Medicine at Mount Sinai), N.S. (Yale University), P. Sklar (Icahn School of Medicine at Mount Sinai), M. State (University of California, San Francisco), P. Sullivan (University of North Carolina), F. Vaccarino (Yale University), S. Weissman (Yale University), K. White (University of Chicago) and P. Zandi (Johns Hopkins University). This work was supported by the Yale/NIDA Neuroproteomics Centre (DA018343-12), by NIA grant AG047270-02, by NIMH grant MH110926, by NIH SIG grants 1S10OD019967-0 and 1S10ODOD018034-01, and by the State of Connecticut, Department of Mental Health & Addiction Services. B.C.C. was supported by a 2014 NARSAD Young Investigator Grant from the Brain & Behavior Research Foundation.
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B.C.C. designed the experiments, performed the experiments, analyzed the data and wrote the manuscript. R.R.K. designed the experiments, analyzed the data and wrote the manuscript. J.E.K. performed the experiments. E.Z.V. performed the experiments. M.P. contributed to tissue and sample processing. A.M.M.S. contributed to tissue and sample processing. T.T.L. designed the experiments and wrote the manuscript. M.B.G. contributed to RNA-seq data generation and provided computational resources. N.S. designed the experiments, contributed to tissue and sample processing, contributed to RNA-seq data generation and wrote the manuscript. A.C.N. designed the experiments and wrote the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Number of proteins detected by highly fractionated proteomics across all regions
Histogram quantifies proteins detected directly by MS/MS (black) versus those detected by the “match between runs” feature (grey) in the fractionated regional samples
Supplementary Figure 2 Number of peptides detected per protein shows no clear inter-regional variability
The average number of peptides detected per protein was 9.2. For simplicity in these histograms, proteins with more than 10 peptides were set to have exactly 10 peptides. The distribution of peptides/protein is similar across all 7 fractionated brain regions
Supplementary Figure 3 Numbers of proteins detected in each single shot sample. The use of “match between runs” results in an approximately 50% increase in the number of proteins identified by single shot proteomics
Proteins directly detected by MS/MS are shown in black, whilst those identified by “match between runs” are in grey. Samples are shown in alphabetical order, and include the data from fractionated brain regions
Supplementary Figure 4 Batch effect identified by sample correlations is corrected by ComBat
A) Clustering of all single shot samples shows a clear batch effect. B) Clustering post correction by ComBat shows correction of the batch effect. C) Technical replicates of a single mixed region control were run evenly spaced throughout the LC-MS/MS runs. Correlation between these technical replicates is strongly improved as a result of batch correction
Supplementary Figure 5 Clustering all samples subjected to MS/MS using proteins significantly differentially expressed between brain regions revealed expected bulk differences between brain regions
This is a fully labelled zoomable version of the main Fig 2B
Supplementary Figure 6 Clustering all proteins significantly differentially expressed between regions reveals consistent patterns of expression that favour region-specific enrichment or region-specific depletion in abundance
Box and whisker plots show region specific patterns of protein expression across 33 gene clusters. The center line indicates the median, limits indicate the IQR, and the whiskers either 1.5* the IQR or the min/max value if it falls within 1.5* the IQR. Expression values from individual samples are shown as dots. Genes belonging to each cluster can be seen in Table 5B and Fig S7
Supplementary Figure 7 Clustering all proteins significantly differentially expressed between regions reveals consistent patterns of expression that favour region-specific enrichment or region-specific depletion in abundance
Heatmap shows expression levels of all differentially expressed genes across all single shot samples. The dendrogram above depicts the clusters of proteins that share patterns of expression across the regions. Cluster 0 is a group of proteins with no shared expression across the regions. Proteins belonging to this cluster are depicted with black arms on the dendrogram, and are not numbered below the heatmap. The heatmap shows that many clusters are dominated by expression changes in the cerebellum compared to other regions
Supplementary Figure 8 Striatally enriched clusters contain interacting proteins with roles in dopaminergic signalling and drug addiction
. A) Network diagram showing proteins from clusters 26 and 31. These clusters are strongly enriched for protein:protein interactions (adj. p value = 1.55E-15). Coloured edges represent different forms of interaction evidence; experimentally determined (pink), coexpressed (black), curated databases (blue) and text mining (green). The node size represents the extent to which the protein structure has been solved. B) Clusters 26 and 31 are significantly enriched for a number of Biological Process ontology terms. C) KEGG pathway analysis shows significant enrichment for expected pathways, including stimulant addiction and dopaminergic synapse
Supplementary Figure 9 Clustering all proteins significantly differentially expressed over developmental period reveals proteins enriched shortly after birth (period 8) and proteins more gently increasing or decreasing in abundance over the time-course
The center line indicates the median, limits indicate the IQR, and the whiskers either 1.5* the IQR or the min/max value if it falls within 1.5* the IQR. Individual samples are shown as dots on the box and whisker plots. Proteins belonging to each cluster can be seen in Table 5
Supplementary Figure 10a RNA vs protein fold-change comparison of all pairs of brain regions
These scatterplots are identically defined to those in Fig 5A except that here genes are labelled to show individual gene names observable at high magnification. A) CBC vs all B) MD vs all C) STR vs all D) AMY vs all E) HIP vs all F) V1C vs all G) DFC vs all. Genes are coloured based on their agreement or disagreement between the RNA and protein measurements; genes for which the protein variability between regions is <2-fold of that reported at the RNA-level were considered consistent (green and grey points). Purple coloured genes are those with consistent direction but variable magnitude of change (≥2-fold) between the regions at the protein and RNA level, while red genes disagree in the direction of change between RNA and protein. Blue and orange genes vary between regions according to protein but not RNA and vice-versa. H) Zoomable, gene labelled versions of the scatter plots in Figure 5c
Supplementary Figure 10b RNA vs protein fold-change comparison of all pairs of brain regions
These scatterplots are identically defined to those in Fig 5A except that here genes are labelled to show individual gene names observable at high magnification. A) CBC vs all B) MD vs all C) STR vs all D) AMY vs all E) HIP vs all F) V1C vs all G) DFC vs all. Genes are coloured based on their agreement or disagreement between the RNA and protein measurements; genes for which the protein variability between regions is <2-fold of that reported at the RNA-level were considered consistent (green and grey points). Purple coloured genes are those with consistent direction but variable magnitude of change (≥2-fold) between the regions at the protein and RNA level, while red genes disagree in the direction of change between RNA and protein. Blue and orange genes vary between regions according to protein but not RNA and vice-versa. H) Zoomable, gene labelled versions of the scatter plots in Figure 5c
Supplementary Figure 10c RNA vs protein fold-change comparison of all pairs of brain regions
These scatterplots are identically defined to those in Fig 5A except that here genes are labelled to show individual gene names observable at high magnification. A) CBC vs all B) MD vs all C) STR vs all D) AMY vs all E) HIP vs all F) V1C vs all G) DFC vs all. Genes are coloured based on their agreement or disagreement between the RNA and protein measurements; genes for which the protein variability between regions is <2-fold of that reported at the RNA-level were considered consistent (green and grey points). Purple coloured genes are those with consistent direction but variable magnitude of change (≥2-fold) between the regions at the protein and RNA level, while red genes disagree in the direction of change between RNA and protein. Blue and orange genes vary between regions according to protein but not RNA and vice-versa. H) Zoomable, gene labelled versions of the scatter plots in Figure 5c
Supplementary Figure 10d RNA vs protein fold-change comparison of all pairs of brain regions
These scatterplots are identically defined to those in Fig 5A except that here genes are labelled to show individual gene names observable at high magnification. A) CBC vs all B) MD vs all C) STR vs all D) AMY vs all E) HIP vs all F) V1C vs all G) DFC vs all. Genes are coloured based on their agreement or disagreement between the RNA and protein measurements; genes for which the protein variability between regions is <2-fold of that reported at the RNA-level were considered consistent (green and grey points). Purple coloured genes are those with consistent direction but variable magnitude of change (≥2-fold) between the regions at the protein and RNA level, while red genes disagree in the direction of change between RNA and protein. Blue and orange genes vary between regions according to protein but not RNA and vice-versa. H) Zoomable, gene labelled versions of the scatter plots in Figure 5c
Supplementary Figure 10e RNA vs protein fold-change comparison of all pairs of brain regions
These scatterplots are identically defined to those in Fig 5A except that here genes are labelled to show individual gene names observable at high magnification. A) CBC vs all B) MD vs all C) STR vs all D) AMY vs all E) HIP vs all F) V1C vs all G) DFC vs all. Genes are coloured based on their agreement or disagreement between the RNA and protein measurements; genes for which the protein variability between regions is <2-fold of that reported at the RNA-level were considered consistent (green and grey points). Purple coloured genes are those with consistent direction but variable magnitude of change (≥2-fold) between the regions at the protein and RNA level, while red genes disagree in the direction of change between RNA and protein. Blue and orange genes vary between regions according to protein but not RNA and vice-versa. H) Zoomable, gene labelled versions of the scatter plots in Figure 5c
Supplementary Figure 10f RNA vs protein fold-change comparison of all pairs of brain regions
These scatterplots are identically defined to those in Fig 5A except that here genes are labelled to show individual gene names observable at high magnification. A) CBC vs all B) MD vs all C) STR vs all D) AMY vs all E) HIP vs all F) V1C vs all G) DFC vs all. Genes are coloured based on their agreement or disagreement between the RNA and protein measurements; genes for which the protein variability between regions is <2-fold of that reported at the RNA-level were considered consistent (green and grey points). Purple coloured genes are those with consistent direction but variable magnitude of change (≥2-fold) between the regions at the protein and RNA level, while red genes disagree in the direction of change between RNA and protein. Blue and orange genes vary between regions according to protein but not RNA and vice-versa. H) Zoomable, gene labelled versions of the scatter plots in Figure 5c
Supplementary Figure 10g RNA vs protein fold-change comparison of all pairs of brain regions
These scatterplots are identically defined to those in Fig 5A except that here genes are labelled to show individual gene names observable at high magnification. A) CBC vs all B) MD vs all C) STR vs all D) AMY vs all E) HIP vs all F) V1C vs all G) DFC vs all. Genes are coloured based on their agreement or disagreement between the RNA and protein measurements; genes for which the protein variability between regions is <2-fold of that reported at the RNA-level were considered consistent (green and grey points). Purple coloured genes are those with consistent direction but variable magnitude of change (≥2-fold) between the regions at the protein and RNA level, while red genes disagree in the direction of change between RNA and protein. Blue and orange genes vary between regions according to protein but not RNA and vice-versa. H) Zoomable, gene labelled versions of the scatter plots in Figure 5c
Supplementary Figure 10h RNA vs protein fold-change comparison of all pairs of brain regions
These scatterplots are identically defined to those in Fig 5A except that here genes are labelled to show individual gene names observable at high magnification. A) CBC vs all B) MD vs all C) STR vs all D) AMY vs all E) HIP vs all F) V1C vs all G) DFC vs all. Genes are coloured based on their agreement or disagreement between the RNA and protein measurements; genes for which the protein variability between regions is <2-fold of that reported at the RNA-level were considered consistent (green and grey points). Purple coloured genes are those with consistent direction but variable magnitude of change (≥2-fold) between the regions at the protein and RNA level, while red genes disagree in the direction of change between RNA and protein. Blue and orange genes vary between regions according to protein but not RNA and vice-versa. H) Zoomable, gene labelled versions of the scatter plots in Figure 5c
Supplementary Figure 11 Summary quantifications of all RNA vs protein fold-change scatter plots
A) Per region-pair counts of the number of genes in each of the colour categories defined in Fig 5A; genes are coloured based on their agreement or disagreement between RNA and protein; genes for which the protein variability between regions was <2-fold of that reported at the RNA-level were considered consistent (green and grey points). Purple coloured genes are those with consistent direction, but variable magnitude, of change between the regions at the protein and RNA level, while red genes disagree in the direction of change between RNA and protein. Blue and orange genes vary between regions according to protein but not RNA and vice-versa. B) Comparison of absolute log2 fold-changes between each region and all others as reported by RNA (blue) and protein (red) show that in general the protein-level fold changes are greater. For example, the DFC plot shows the distribution, over all genes, of fold changes by RNA and by protein of the DFC with each of the 6 other regions. The center line indicates the median, limits indicate the IQR, and the whiskers either 1.5* the IQR or the min/max value if it falls within 1.5* the IQR. Outlying genes are not shown in this plot to highlight the increased median and 75th percentile of the protein fold-change distributions
Supplementary Figure 12 DFC/V1C comparison in detail
A) The scatterplot is identical to the corresponding panel in Fig 5A, except that individual gene names are labelled. Proteins used for validation in parts C & D are highlighted with pink font. B) Protein-protein interaction network, produced by STRING (medium stringency), of the genes upregulated in DFC in protein only (blue points, right hand side Fig S12A). Proteins used for validation in parts C & D are highlighted with pink font/circles. C) Immunoblotting of the 5 adult DFC and V1C samples shows enrichment of GRM2/3, CNR1 and PDE4D in the DFC over the V1C. Note that the blots shown in this figure are cropped images, and that CNR1 and NTRK3 labelling were performed on the same cut membrane, and thus have the same GAPDH control. D) Quantification of the immunoblots (values normalized to GAPDH) shows significant enrichment of GRM2/3, CNR1 & PDE4D in DFC over V1C by twotailed paired student’s T-test (n = 5 biological replicates per group, d.f. = 4, t = 5.842, 7.006, 3.067 respectively)
Supplementary Figure 13 Summary quantifications of all fold-change scatter plots for human vs mouse
Per region-pair counts of the number of genes in each of the colour categories defined in Fig 6B; genes are coloured based on their agreement or disagreement between mouse and human; genes for which the human variability between regions was within 2-fold of that reported for mouse were considered consistent (green and grey points). Purple coloured genes are those with consistent direction, but variable magnitude, of change between the regions in human vs mouse, while red genes disagree even in the direction of change between the species. Blue and orange genes vary between human regions but not mouse and vice-versa
Supplementary Figure 14a Human protein vs mouse protein fold-change comparison of all pairs of brain regions
The scatterplots are identically defined to those in Fig 6B except that here only significantly differentially expressed genes from the human are included and genes are labelled to show individual gene names. A) CBC vs all. B) MD vs all. C) STR vs all. D) HIP vs all. E) DFC vs all
Supplementary Figure 14b Human protein vs mouse protein fold-change comparison of all pairs of brain regions
The scatterplots are identically defined to those in Fig 6B except that here only significantly differentially expressed genes from the human are included and genes are labelled to show individual gene names. A) CBC vs all. B) MD vs all. C) STR vs all. D) HIP vs all. E) DFC vs all
Supplementary Figure 14c Human protein vs mouse protein fold-change comparison of all pairs of brain regions
The scatterplots are identically defined to those in Fig 6B except that here only significantly differentially expressed genes from the human are included and genes are labelled to show individual gene names. A) CBC vs all. B) MD vs all. C) STR vs all. D) HIP vs all. E) DFC vs all
Supplementary Figure 14d Human protein vs mouse protein fold-change comparison of all pairs of brain regions
The scatterplots are identically defined to those in Fig 6B except that here only significantly differentially expressed genes from the human are included and genes are labelled to show individual gene names. A) CBC vs all. B) MD vs all. C) STR vs all. D) HIP vs all. E) DFC vs all
Supplementary Figure 14e Human protein vs mouse protein fold-change comparison of all pairs of brain regions
The scatterplots are identically defined to those in Fig 6B except that here only significantly differentially expressed genes from the human are included and genes are labelled to show individual gene names. A) CBC vs all. B) MD vs all. C) STR vs all. D) HIP vs all. E) DFC vs all
Supplementary Figure 15 Un-cropped versions of the immunoblots in Figure S12
Note that all GADPH blots were visualised using Licor 800 antibodies, and only the 25 kDa ladder band is visible at this wavelength. A) mGlur2/3 and the corresponding GADPH blot. B) CNR1 and the corresponding GAPDH control. This blot was then trimmed and re-probed for NTRK3. C) PDE4D immunoblot and corresponding GAPDH control. D) NTRK3 immunoblot. See B) for GAPDH control
Supplementary Information
Supplementary Text and Figures
Supplementary Figures 1–15 and Supplementary Table 11
Supplementary Table 1
Metadata for all 77 BrainSpan samples subjected to MS/MS for this study.
Supplementary Table 2
Peptide-level data obtained from heavily fractionated per-region MS/MS.
Supplementary Table 3
Protein-level summary of the fractionated per-region and single-shot MS/MS.
Supplementary Table 4
Label-free protein quantification (LFQ) of all single-shot samples.
Supplementary Table 5
Results of the proteomic spatiotemporal differential expression analysis.
Supplementary Table 6
Protein and RNA expression data for genes expressed in both datasets.
Supplementary Table 7
Inter-regional protein and RNA abundance and differential expression summary.
Supplementary Table 8
Summary of the RNA vs. protein differential consistency of each gene in accordance with the definitions introduced in Fig. 5.
Supplementary Table 9
Complete ontology and gene-set enrichment analysis results consistent with the definitions introduced in Fig. 5.
Supplementary Table 10
Inter-regional human and mouse protein abundance summary.
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Carlyle, B.C., Kitchen, R.R., Kanyo, J.E. et al. A multiregional proteomic survey of the postnatal human brain. Nat Neurosci 20, 1787–1795 (2017). https://doi.org/10.1038/s41593-017-0011-2
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DOI: https://doi.org/10.1038/s41593-017-0011-2
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