# Differential Proteomic Analysis of _Listeria monocytogenes_ during High-Pressure Processing
## 3 Results
### Machine Learning of Grouping DEPs  
This section is on producing groups of 79 proteins with a high degree of similarity within each group and a low degree of similarity between groups by using an unsupervised \(k\)-means clustering learning algorithm (Figure 1). Four clusters are the optimal number of clusters after Gap Statistic analysis, where the smallest \(k\) was determined by \(Gap(k)\geq Gap(k+1)-s_{k+1}\) (Figure S3). After determining the optimal number of optimized clusters \(k\) = 4, the \(k\)-means was applied to divide 79 proteins into 4 clusters (Table S2). The detailed characteristics of grouping DEPs are listed in Table S3, where four different evaluations are group 9G, 9 GE, 3G and 3 GE. The 48 upregulated DEPs were classified into four groups according to the hierarchical clustering heat maps: UI: 8 DEPs including rpsT, rpsL, rpsM, rpmF2, rpmA, rplX, rpsU and infC; UII: dapF; UIII: 10 DEPs including luxS, rpsJ, rplO, rplF, rpmD, rplI, rpsI, rpsP, rpsG and rpsS; UIV: the other 29 DEPs (Figure 2b). The 8 DEPs of the group UI exhibited apparent up-regulated (>3.0 log\({}_{2}\) (fold change)). rpsU, infC and rplX was upregulated by 4.0 folds under H4/C treatments. dapF (group UII) was upregulated in decreasing order (1.78 for H2/C and 1.28 for H3/C) but for H4/C was slightly downregulated (\(-\)0.41). Except for rpsI, rpsJ and luxS, 7 out of 10 DEPs in the group UIII revealed an increasing order of upregulation--mostly within the range from 2 to 3 log\({}_{2}\) (fold change). All the 29 DEPs of the group UIV showed lower levels (<2.0) of upregulation except for tall (2.64) and rplR (2.10) under H4/C treatment  
On the other hand, the 48 upregulated DEPs were classified into 2 clusters according to k-means clustering: 16 DEPs for cluster 1 (HUR) and 32 DEPs (UR) for cluster 2 (Figure 2b and Table S2). All treatments of 16 DEPs exceeded 2.0 log\({}_{2}\) (fold change) except for rpmD (1.97) and rplO (1.89) under H2/C treatments. The DEPs of HUR cluster were the same as the hierarchical clustering group UI and UIII excluding luxS and rpsJ. UR (cluster 2) had 32 DEPs in the hierarchical clustering group UII, UIV, luxS and rpsJ. There are four different evaluations, 9G, 9 GE, 3G and 3 GE groups, depending on each repetition and its mean values. All four clusters (HUR, UR, DR and HDR) had the same numbers of DEPs under the four evaluations except for rplO. rplO represented clusters 1 and 2 under 9G/9 GE/3G, and 3 GE evaluations, respectively. The DEPs of cluster 1 (HUR) in group 3G had a greater than 2.0 log\({}_{2}\) fold change under both H3/C and H4/C treatments, whereas the DEPs, including rplO, of cluster 2 at 3 GE had a less than 1.0 log\({}_{2}\) fold change. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Functional** & & **Upregulation** & **Downregulation** \\
**Characterization** & & \\ \hline Regulation of cell septum & ko04812 Cytoskeleton proteins & spoVG12  & \\ \hline Exosome & ko04147 Regulation of & adk, tpiA12  \\  & Exosome (2) & adk, tpiA12  \\ \hline
**Organisal Systems (OS)** & & \\ \hline Aging & & \\ \hline  & 04212 Longevity regulating & clpP \\  & pathway—worm & \\ \hline
**Human Diseases (HD)** & & \\ \hline Infectious disease: bacterial & 05150 _Staphylococcus aureus_ & dltC2  \\  & infection & & \\ \hline Drug resistance: & 01502 Vancomycin resistance & Alr2  \\ \hline  & 01503 Cationic antimicrobial & & dltC2  \\  & peptide (CAMP) resistance & & dltC2  \\ \hline \hline \end{tabular}  
* 1: DEP only detected in H2/C.[ENDFOOTNOTE]  
\end{table} 
Table 1: _Cont._The 31 downregulated DEPs were classified into four groups according to the hierarchical clustering heat maps: DI: rpmG1; DII: dltC and lmo1501; DIII: rbfA; DIV: the other 27 DEPs (Figure 2b). rpmG1 (in group DI) was notably different from other downregulated DEPs that exhibited apparent downregulated in H2/C and H3/C more than \(-4\) but showed slightly upregulated in H4/C. The dltC showed in a dose-dependent fashion that H3/C and H4/C had a \(-3.71\) and \(-5.20\log_{2}\) fold change, respectively.