# Effect of Mechanical Damage in Green-Making Process on Aroma of Rougui Tea
## 3 Results
### Proteome Profiling of Proteins Differentially Expressed in Response to Green-Making Process  
SY, LY7 and CK7 were extracted and qualitatively quantified by the TMT labeling technique to explore the mechanism of influence of the green-making process on RGT's aroma metabolism from the perspective of proteomics. A total of 623,142 secondary spectra were identified in all samples, among which 36,759 peptide spectra and 29,159 unique peptide spectra were identified. A total of 19,376 peptides were identified, including 16,650 unique peptides and 5242 proteins. The relative molecular weight distribution statistics of the proteins are shown in Figure 2A; 49.93% of the proteins were 0-50 kDa, 39.97% were 51-100 kDa and 10.10% were greater than 100 kDa. Most of the peptide lengths in the proteins were distributed among 6-13 amino acid residues, accounting for 73.41% of the total peptide number (Figure 2B). The proportions of proteins with peptide sequence coverage below 10%, 10-20%, 20-30%, 30-40% and 40-100% were 50.32%, 30.14%, 12.57%, 4.63% and 2.34%, respectively (Figure 2C). The proportion of proteins with peptide sequence coverage below 20% accounted for 80.46% of the total proteins. The number distribution of the peptides contained in the proteins identified is shown in Figure 2D. The number of proteins containing one to five peptide segments was 3377, accounting for 70.49%; the number of peptides containing 6 to 10 peptide segments was 1020, accounting for 21.29%; and the number of peptides containing 11 or more peptide segments was 394, accounting for 8.22%, indicating that the number of peptides contained in most proteins was within five, followed by 6-10. In this study, a total of 5242 proteins were identified, which were screened according to the conditions of a more than 1.2 times difference and a \(p\)-value less than 0.05. The results showed that there were 635 DEPs between SY and CK7, and the number of upregulated and downregulated proteins was 378 and 257, respectively. There were 729 DEPs between SY and LY7, and the number of upregulated and downregulated proteins was 495 and 234, respectively. There were 385 DEPs between CK7 and LY7, and the number of upregulated and downregulated proteins was divided into 270 and 115 (Figure 3A-D), which was consistent with the metabolic data. Long-term green-making can significantly change the metabolic activity of substances in fresh tea leaves, improve the intensity of protein synthesis and promote the production of metabolic substances. In living organisms, proteins usually do not perform their functions independently, but many different proteins cooperate with each other to complete a biological function or participate in a metabolic pathway. The significant enrichment of pathways can identify the major biochemical metabolism and signal transduction pathways involved in DEPs. The KEGG enrichment analysis (Figure 3E,F) showed that the differential proteins between SY, LY7 and CK7 were involved in metabolic pathways including flavonoid biosynthesis, alpha-linolenic acid metabolism, linoleic acid metabolism and fatty acid metabolism, the biosynthesis of unsaturated fatty acids, glyoxylate and dicarboxylate metabolism, carotenoid biosynthesis, phenylalanine metabolism and terpenoid backbone biosynthesis. Compared with CK7, DEPs in LY7 involved more significant aroma metabolic pathways and synthesized more abundant aroma compounds. The biological metabolism of fatty acids can produce a large number of aroma precursors, which participate in the metabolic pathways of linoleic acid and \(\alpha\)-linolenic acid, and these unsaturated fatty acids are oxidized and degraded to form rich aliphatic aroma compounds; one of the branch pathways of \(\alpha\)-linolenic acid is related to the formation of jasmonic acid, and the accumulation of jasmonic acid promotes the expression of terpenoid synthesis genes and the synthesis of terpenes [37].