# Proteomic Landscape of the Mature Roots in a Rubber-Producing Grass _Taraxacum Kok-saghyz_
## 2 Results
### Pathway Analysis of the Identified Proteins in 6M TKS Roots  
To gain insight into the functional categories of the 3592 proteins (including the 231 and 3545 proteins identified by 2-DE and shotgun, respectively), we performed gene ontology (GO) classification and the enriched outputs of their biological process, cellular component and molecular function are presented (Figure 5). The GO enrichment revealed that many proteins were localized in membrane-bounded organelle, and they were mainly involved in metabolic process with catalytic activity or binding ability (Figure S3; Table S4). At the biological process level, 13 main processes were detected. Among them, the largest amount containing 1520 proteins were involved in the metabolic process, followed by 1033 proteins involved in the cellular process and 695 proteins in the single-organism process. A total of 302 proteins were considered to respond to external stimulus. At subcellular level, nine components were observed. Among them, almost half including 866 proteins were localized to the cell part. Many proteins were localized into organelle membrane and macromolecular complex. In molecular function classification, seven pathways were determined. Among them, the most portion including 1688 proteins were taken part into catalytic activity, followed by 1325 proteins with binding ability. There are 107 proteins with transporter activity and 101 proteins with structural molecule activity (Figure 5; Table S4). These different GO term distributions in TKS roots are related to their biological functions and indicate some important biological processes for secondary metabolite in the 6M TKS roots. To further investigate the biological functions of the identified 3592 proteins, KEGG pathway analysis was performed using the BLAST2 GO program. These proteins were clustered into 19 main pathways (Figure 6A), including translation process (616 proteins), carbohydrate metabolism (583 proteins), folding, sorting and degradation (440 proteins), global and overview metabolism (409 proteins), amino acid metabolism (380 proteins), transport and catabolism (342 proteins), lipid metabolism (308 proteins), etc. These results demonstrated that the main proteins in TKS roots were involved in translation and carbohydrate metabolism. Furthermore, 129 sub-pathways were enriched in KEGG annotation (Table S5), and the top 20 sub-pathways are highlighted (Figure 6B). Among them, the most abundant pathway containing 240 proteins is carbon metabolism. The second contains 224 proteins with ribosome activity. The third sub-pathway has 213 proteins for biosynthesis of amino acids, followed by 183 proteins for endocytosis, 182 proteins in the spliceosome, and 172 proteins in the endoplasmic reticulum. Proteins involved in starch and sucrose metabolism (164 proteins), glycolysis (121 proteins), phenylpropanol biosynthesis (119 proteins), and amino sugar and nucleotide sugar metabolism (116 proteins), are also enriched in these KEGG sub-pathways (Table S5). These proteins are crucial for biosynthesis of isoprenoids in many plants, especially in natural rubber-producing plants [16]. Figure 4: High-throughput proteomics analysis of the 6M TKS roots. Three shotgun proteomics were performed to determine the large-scale protein profiles in the 6M roots, and finally identify 3545 shared proteins in the three experiments (**A**) The blue, yellow, and pink circles represent for the first, second, and third biological replicates. Among these proteins, 184 proteins could also be detected from the 2-DE gel (**B**). The pink area represents for the 184 shared proteins that were identified by both the 2-DE and shotgun based proteomic methods. Figure 5: GO classification of the identified proteins. The 3592 proteins identified from the 6M roots by both shotgun and 2-DE methods were performed GO analysis and these proteins were classified into 3 main categories including biological process, cellular component and molecular function. The number of proteins denotes those with GO annotations.