In this study, brain tissue proteins from four frontal and four temporal cortex were collected. The detailed clinical information for 8 patients involved in this study is listed in Table 1. The proteomic profiling was performed based on brain tissue (Brodmann’s area 38 or 10). All patients were diagnosed with cerebral contusions and hemorrhage and underwent an operation of intracerebral hematoma clearance for treatment.
Proteomics profile of frontal and temporal cortex
In the experiment, a total number of 2127 protein groups were identified in either frontal or temporal cortex tissues (Fig. 2a). The protein group information is listed in Supplemental Table 1A. Of which, 1838 proteins could be quantitated in all four frontal cortex samples with three technical replicates and were used for technical variation analysis. The detailed protein information is listed in Supplemental Table 1B. The median of technical CVs was 6.95%, and 95% of the identifications (1746) showed a technical CV lower than 22.4% (Fig. 2b). In the temporal cortex proteome, a total of 2124 proteins were identified. Of which, 1839 proteins could be quantified (Fig. 2a). The information is shown in Supplemental Table 1C. The median technical CVs were 9.36%, and 95% of the identifications (1747) showed a technical CV lower than 28.8%, which was slightly higher than the CV in the frontal cortex proteome (Fig. 2c). In the following data analysis, the proteins with the highest 5% technical CVs were excluded to minimize the interference of technical variations. Finally, a total number of 1746/1747 proteins in frontal/temporal were used for an inter-individual analysis.
Inter-individual variations in the frontal and temporal cortex proteome
In the frontal/temporal lobe proteome, the median inter-individual CVs from the analysis of 1746/1747 proteins were 11.3%/11.8%. The inter-individual CVs of 95% identifications in the frontal/temporal cortex samples were lower than 29.9/32.5 (Fig. 2d, e). The inter-individual CVs of the two brain tissue proteomes were similar to each other. Moreover, 1709 proteins were both identified and quantitated in the frontal and temporal lobe tissues (Fig. 2a). The detailed information is shown in Supplement Table 2A. The median inter-individual CVs were 11.3 and 11.8 respectively; the CVs of the two tissues were similar (Supplemental Figure 1A). Then the inter-individual CVs of 1709 frontal and temporal lobe proteins were plotted against each other. As shown in Supplemental Figure 1B, approximately most of the proteins had similar inter-individual variations between the two brain tissues. These results demonstrated that frontal and temporal lobe proteomes are both stable to some extent.
Comparison of technical and individual variations
To view the correlation of technical and individual variations in the brain tissues, the CVs were plotted against each other. As shown in Supplemental Figure 2 A and B, proteins that demonstrated similar technical and inter-individual CVs were grouped close to the 45° line. In both frontal and temporal lobe tissues, most proteins exhibited larger inter-individual CV than a technical CV. Besides, compared with the median technical CVs 6.95%/9.36% observed between the different sequential runs, the median inter-individual CVs 11.3%/11.8% was larger. Those results indicated that the technical variation contributed less contribution to the total variation than the individual variation.
Analysis of differentially expressed proteins
Differentially expressed proteins (DEPs) between the frontal cortex and temporal cortex were first assessed by Student’s test (p<0.05), and 90 DEPs are showed in Supplementary Table 2D. Correlation coefficient plot, heatmap, and principal component analysis (Fig. 3a–c) were used to investigate the dynamics and reproducibility of DEPs between frontal and temporal cortex. The frontal and temporal cortex can be differentiated into different clusters using PCA. Based on the p-value and 1.3-fold change, compared to proteins expressed in the frontal cortex, 14 proteins were screened highly expressed in the temporal cortex, including MAPT, SNCG, ATP5IF1, GAP43, HSPE1, STMN1, NDUFS6, LDHB, SNCB, NDUFA7, MRPS36, NDUFA, EPDR1, CISD1, and RALA. In addition, compared to proteins expressed in the frontal cortex, 14 proteins including EDC4, NIT2, VWF, ASTN1, TGM2, SSB, CLU, HBA1, STOM, CRP, LRG1, SAA2, S100A4, and VTN were a low expression in the temporal cortex (Fig. 3d). The expression pattern of these upregulated proteins in the temporal cortex are shown in Fig. 4A1–A14. The expression pattern of those downregulated proteins in the temporal cortex is shown in Fig. 4B1–B14.
Bioinformatics and functional enrichment analysis
Functional annotation of frontal and temporal cortex proteins by Ingenuity Pathway Analysis (IPA)
To further investigate the biological pathways, functions, and networks in the frontal and temporal tissues, we analyzed the identified proteins with the IPA software. The top ten significant IPA canonical pathways are listed in Fig. 5a. Among them, mitochondrial dysfunction was the most enriched pathway. Mitochondria are the primary consumers of oxygen in cells, responsible for ATP production and oxidative phosphorylation. Mitochondrial dysfunction may bring neurons a high risk of damage or death. In this experiment, numbers of identified proteins were categorized in mitochondrial dysfunction, which adds the evidence that brain function is importantly based on the normal function of mitochondria. Changes in brain proteins possibly lead to mitochondria dysfunction. The other pathways included oxidative phosphorylation, clathrin-mediated endocytosis signaling, and EIF2 signaling. Besides, when the physiological system development and function of these proteins were analyzed, the top function was predicated as nervous system development and function, and 532 of 1709 identified proteins were involved in this function. Moreover, when the disease and function annotations for the identified proteins were performed, the top results in a neurological disease (Fig. 5b), and 370 proteins were connected to this annotation. These results together show that proteins related to neurological function were enriched in brain tissue.
The frontal cortex, along with the other associational cortical areas, is involved in cognitive control processes. The temporal lobe is essential for visual memories and language comprehension. Not only do the protein constituents of the frontal and temporal cortex provide useful information regarding the physiology and pathology of the central nervous system, but they also offer valuable clues for neurological diseases. Many molecules were involved in the neurological diseases by IPA functional analysis (Supplemental Table 3). These diseases include Alzheimer’s disease, Huntington’s disease, and glioblastoma. According to IPA analysis, the top related disease was movement disorder, which had forty-seven categorized proteins. Most of these proteins are related to the inflammatory response, including CP, APOE5, and HP. For acute movement disorders in children, autoimmune and inflammatory disorders are the most common causes [15].
Quantified proteins were separated into two parts based on p-value including 90 DEPs (p<0.05) and 1619 unchanged proteins (p≥0.05) between the frontal and temporal cortex. In order to further understand the functional differences between the two parts, functional enrichment analysis and KEGG signaling pathway analysis were carried out, respectively. As a result, the main biological processes enrichment of unchanged proteins are regulated exocytosis, axon guidance, vesicle-mediated transport, generation of precursor metabolites and energy, regulation of vesicle-mediated transport, cofactor metabolic process, trans-synapses, etc. (Fig. 6a, Supplemental Table 4A). The main biological processes enrichment of DEPs are regulated exocytosis, propanoate metabolism, autophagy, positive regulation of viral genome replication, protein folding, etc. (Fig. 6b, Supplemental Table 4B).
Pathway analysis of unchanged proteins and DEPs
To gain a deeper understanding of the pathway of unchanged proteins and DEPs between the frontal cortex and temporal cortex, we performed a KEGG pathway analysis.
The main KEGG signaling pathway of unchanged proteins are oxidative phosphorylation, Huntington’s disease, carbon metabolism, Parkinson’s disease, biosynthesis of antibiotics, Alzheimer’s disease, citrate cycle (TCA cycle), metabolic pathways, ribosome, endocytosis, non-alcoholic fatty liver disease (NAFLD), synaptic vesicle cycle, glutamatergic synapse, GABAergic synapse, dopaminergic synapse, etc. (Fig. 6c, Supplemental Table 4C). The main KEGG signaling pathways of DEPs are propanoate metabolism and oxidative phosphorylation (Fig. 6d, Supplemental Table 4D).
Functional analysis of part of upregulated and downregulated proteins in temporal cortex
Statistical analysis showed that compared to protein expression in the frontal cortex, a total number of 28 proteins were significantly upregulated or downregulated in the temporal cortex.
Microtubule-associated protein tau (MAPT, TAU) is one of the major microtubule-associated proteins in the vertebrate nervous system which promotes microtubule assembly and stability and might be involved in the establishment and maintenance of neuronal polarity [16]. Tau is specifically abundant in neurons of the central nervous system [17]. Tau is also closely associated with Alzheimer’s disease occurrence and progression [18]. Tau usually with multiple post-translational modifications (PTM) including phosphorylation, acetylation, disulfide bond, N-linked or O-linked glycosylation, isopeptide bond, methylation, and ubiquitination conjugation, and these PTMs as well as tau aggregation are closely related to its function and Alzheimer’s disease (AD). Tau protein is a component of the paired helical filaments associated with AD. Tau is also the main constituent of the intraneuronal neurofibrillary tangles (NFTs), which is one of the two main hallmarks of the disease in searching for disease-modifying therapies for AD [19]. Most importantly, tau protein is one of the targets and tau-targeting therapies for AD including educing tau expression, tau protein modifications inhibitors, tau aggregation inhibitors, and tau immunotherapies. Currently, the majority of tau-targeting therapies in clinical trials are immunotherapies, which have shown promise in numerous preclinical studies. Other approaches have been discontinued because of toxicity and/or lack of efficacy. Tau mRNA expression at the gene level had no obvious regional variability in a previous study [20]; however, in this study, a higher abundance of MAPT was found in the temporal cortex. As a result, the regional distribution of MAPT protein in the brain may be very important, especially for Alzheimer’s disease studies. From the results of this study, compared to the frontal cortex, tau protein is higher expressed in the temporal cortex.
Gamma-synuclein (SNCG) is a phosphoprotein highly expressed in the brain, particularly in the substantia nigra. SNCG plays a role in neurofilament network integrity. May be involved in modulating axonal architecture during development and in the adult.
Neuromodulin (GAP43) is an important component of the presynaptic terminal and axon. Alterations in cerebrospinal fluid levels of GAP43 may aid in the clinical diagnosis of frontotemporal dementia [21].
Stathmin-1 (STMN1) performs an important function in the regulation of microtubule dynamics and neurite elongation. For patients with intractable temporal lobe epilepsy, the expression of STMN1 was significantly decreased in the neuronal membrane and cytoplasm than in healthy controls [22].
The S100 proteins family are associated with anti-inflammatory processes and are always related to brain injury [23]. The protein S100A4 belongs to S100 proteins family was upregulated during brain injury and considered a therapeutic target in neuronal survival and neuroprotection via JAK/STAT and the IL-10 receptor [24]. Overexpression of S100A4 modulates varieties of antioxidant enzymes and neuroprotective genes. In this study, the S100A4 protein had significantly higher expression in the frontal lobe. In a previous study, the expression of S100A4 was also weak in the hippocampus and the temporal cortex in the human fetal brain [25].
Clusterin (CLU) functions as an extracellular chaperone that prevents aggregation of non-native proteins, prevents stress-induced aggregation of blood plasma protein s[26], and inhibits the formation of amyloid fibrils by APP, APOC2, B2M, CALCA, CSN3, SNCA, and aggregation-prone LYZ variants in vitro [27, 28]. CLU is an important element in the control of extracellular protein misfolding including Alzheimer’s beta-peptide.
C-reactive protein (CRP) displays several functions associated with host defense: it promotes agglutination, bacterial capsular swelling, phagocytosis, and complement fixation through its calcium-dependent binding to phosphorylcholine. The concentration of CRP in plasma increases greatly during the acute phase response to tissue injury, infection, or other inflammatory stimuli.
Astrotactin-1 (ASTN1) is located on the neuronal cell surface that mediates neuron migration [29]. These may help to understand the difference of the function in the two brain regions.