3.1. Identification of DEPs and DEPPs in Early and Late stages of TBI
In proteome profiling, 4586 protein groups were identified. The cutoff of fold-change was set as more than 1.2 or less than 0.83. Totally 3423 proteins were quantified, where 123 proteins are high-expressed and 49 proteins are low-expressed in group 1d/C, and 109 proteins are high-expressed and 9 proteins are low-expressed in group 7d/C, and 129 proteins are high-expressed and 88 proteins are low-expressed in group 7d/1d when compared to the control sample (Figure 1A).
The TMT labeling, enrichment of Phos affinity, and high-resolution LC-MS/MS analysis were applied to quantitative phosphoproteomics analysis. Among the 2229 protein groups, 5961 phosphorylation sites were recognized, where 4095 sites in 1656 proteins were quantified. The cutoff of fold-change was set as more than 1.2 or less than 0.83. Among the quantified proteins, 315 proteins are high-expressed and 453 proteins are low-expressed in group 7d/C, 360 proteins are high-expressed and 738 proteins are low-expressed in group 1d/C, and 682 proteins are high-expressed and 508 proteins are low-expressed in group 7d/1d when compared to the control sample (Figure 1B).
3.2. Functional Enrichment-based Clustering Analyses of DEPs and DEPPs at Early and Late stage in TBI
The functional enrichment analyses based on DEPs (Figure 1C) or DEPPs (Figure 1D), including biological process, molecular function, cellular component, and KEGG pathway analyses, were clustered to compare the difference in group 1d/C, 7d/1d, and 7d/C.
Based on DEPs, the different biological processes in group 1d/C are multi-organism process, regulation of hydrolase activity, and acute-phase response, etc, while the different biological processes in group 7d/C are neuron projection regeneration, regulation of protein modification, etc, and the different biological processes in 7d/1d are regulation of cell growth and cholesterol transport, etc, suggesting a process from acute-phase response to neuron repairing after brain trauma. The different molecular functions in group 1d/C are serine-type endopeptidase inhibitor activity, phospholipase inhibitor activity, etc, while the different molecular functions in group 7d/C are phosphatidylcholine-sterol O-acyltransferase activator activity, etc, suggesting a modification of phosphorylation after brain trauma. The different cellular components in group 1d/C are extracellular vesicles and exosomes, etc, while the different cellular components in group 7d/C are fibrinogen and chylomicron, etc, suggesting a transformation from early cell-cell communications to late coagulation.
Based on DEPPs, the different biological processes in group 1d/C are the establishment of protein localization to the plasma membrane, cell migration, actin filament bundle assembly, etc, while the different biological processes in group 7d/C are neuron projection, signaling, etc, and the different biological processes in group 7d/1d are negative regulation of MAPK activity, etc, suggesting an early cell-cell communication that mediates signalings to result in neuron repairment. The different cellular components in group 1d/C are leading edge membrane, etc, while the different cellular components in group 7d/C are adhering junction, synapse, etc. The different KEGG pathways in group 1d/C are VEGF signaling, oxytocin signaling, Rap1 signaling, etc, while the different KEGG pathways in group 7d/C are calcium signaling, NOD-like receptor signaling, retrograde endocannabinoid signaling, etc, and the different KEGG pathway in group 7d/1d is insulin resistance.
3.3. Motif Analysis of The Phosphosites
We identified 34 conserved motifs based on serine (S) phosphosites, 8 conserved motifs based on threonine (T) phosphosites, and 3 conserved motifs based on tyrosine (Y) phosphosites (Figure 2A). In particular, motif [xxxxxxxxxx_pS_PExxxxxxxx], [xxxxxxxRRx_pS_xxxxxxxxxx], [xxxxxxxxxx_pS_DxExxxxxxx], [xxxxxxxxxx_pS_EEExxxxxxx], [xxxxxxxxxx_pS_xDExxxxxxx], [xxxxxxxRxx_pS_Lxxxxxxxxx], and [xxxxxxxxxx_pT_SPxxxxxxxx] were strikingly conserved with motif score > 30. Among these motifs, [xxxxxxxxxx_pS_EEExxxxxxx] and [xxxxxxxxxx_pT_SPxxxxxxxx] are most significant with fold change > 10. The motif [xxxxxxxxxx_pS_EEExxxxxxx] was identified as the Casein Kinase II substrate motif[12], while the motif [xxxxxxxxxx_pT_SPxxxxxxxx] was a novel motif.
Further, the heatmaps were depicted to illustrate the enrichment or depletion of specific amino acids around the phosphosites of the serine (S) (Figure 2B), threonine (T) (Figure 2C), and tyrosine (Y) (Figure 2D). The amino acids asparticacid (D), glutamicacid (E), lysine (K), and arginine (R) tended to be present in the proximity of serine phosphosites. The amino acids asparticacid (D), glutamicacid (E), proline (P), serine (S), and arginine (R) tended to be present in the proximity of threonine phosphosites. The amino acids asparticacid (D), glutamicacid (E), lysine (K), valine (V), and arginine (R) tended to be present in the proximity of tyrosine phosphosites. Besides, Arginine (R) was increasingly presented at the sites around the serine phosphosites and threonine phosphosites but was strikingly depleted at +1 and +2 positions (Figure 2B&C). Interestingly, Arginine (R) was inclined to be presented at the sites surrounding the tyrosine phosphosites while being depleted at -1 and +1 positions (Figure 2D). Besides, lysine (K) was greatly presented at the sites surrounding the tyrosine phosphosites but was strikingly depleted at +1 and +3 positions (Figure 2D). Considering that we identified the different molecular functions in group 1d/C are serine-type endopeptidase inhibitor activity, phospholipase inhibitor activity, while the different molecular functions in group 7d/C are phosphatidylcholine-sterol O-acyltransferase activator activity, these special amino acids near the phosphosites may reflect the preferable enzymes that catalyze phosphorylation after the brain trauma.
3.4. Common Cross-talk Pathway/Process and Protein-protein Interacting Analyses of DEPs and DEPPs at Early and Late stage in TBI
The common cross-talk pathway/process enrichment analysis of DEPPs (Figure 3A) and DEPs (Figure 3B), and protein-protein interaction enrichment analysis of DEPPs (Figure 3C) and DEPs (Figure 3D) were performed to further identify the connections and similarities among different functional clusters.
For cross-talk pathway and process enrichment analysis of DEPPs (Figure 3A), the identified pathways and processes were clustered according to their similarity functions. The top 20 clusters further clustered into four separate super-clusters in group 1d/C and five separate super-clusters in group 7d/C. For cross-talk pathway and process enrichment analysis of DEPs (Figure 3B), the top 20 clusters further clustered into a super-cluster in group 1d/C and three separate super-clusters in group 7d/C.
For protein-protein interaction enrichment analysis of DEPPs (Figure 3C), the interactive proteins clustered into three main clusters in group 1d/C, where PCE/CE pathway, WNT signaling, chemical synapse transmission, small GTPase mediated signaling, and RAS signaling are important. In group 7d/C, the main cluster is the synapse and postsynapse-related proteins, suggesting the importance of chemical synapse transmission. In addition, in group 7d/C, the cluster regulation of NMDA receptors and neuron projection is also important. For protein-protein interaction enrichment analysis of DEPs (Figure 3D), in group 1d/C, the main clusters are post-translation protein phosphorylation, platelet degranulation, acute response to elevated platelet cytosolic Ca2+, and respiratory complexⅠbiogenesis, suggesting early protein phosphorylation and acute-phase response. In group 7d/C, the main clusters are post-translational protein phosphorylation, platelet degranulation, regulation of insulin-like growth factor (IGF) transport and uptake.
3.5. Special Cross-talk Pathway/Process and Protein-protein Interacting Analyses of DEPs and DEPPs at Early and Late stage in TBI
The special cross-talk pathway/process and protein-protein interacting analyses of DEPPs (Figure 4A) and DEPs (Figure 4B) were performed to further identify the in-depth connections behind the cross-talk pathway and process.
For DEPPs, the special cross-talk pathway/processes that are only presented in group 1d/C (involving 468 proteins) are cell part morphogenesis, membrane trafficking, synapse organization, etc, where the main protein-protein interactions are involved in UCH proteinases, mitotic prometaphase, vesicle docking, etc. The special cross-talk pathway/processes that are only presented in group 7d/C (involving 191 proteins) are the regulation of cell morphogenesis, actin filament-based process, small GTPase mediated signal transduction, etc. The special cross-talk pathway/processes that are both presented in group 1d/C and 7d/C (involving 505 proteins) are neuron projection morphogenesis, modulation of chemical synaptic transmission, etc, where the main protein-protein interactions are involved in localization with/to the membrane, modulation of chemical synaptic transduction, signaling by RhoGTPase, cellular responses to stress, etc.
For DEPs, the special cross-talk pathway/processes that are only presented in group 1d/C (involving 125 proteins) are regulation of the microtubule-based process, supramolecular fiber organization, neutrophil degranulation, etc, where the main protein-protein interaction is involved in respiratory chain complexⅠbiogenesis. The special cross-talk pathway/processes that are only presented in group 7d/C (involving 71 proteins) are cell junction organization, negative regulation of cellular component organization, vacuole organization, etc. The special cross-talk pathway/processes that are both presented in group 1d/C and 7d/C (involving 47 proteins) are platelet degranulation, complement and coagulation cascades, negative regulation of hydrolase activity, etc, where the main protein-protein interactions are involved in post-translational protein phosphorylation, platelet degranulation, regulation of insulin-like growth factor (IGF) transport and uptake.