Identification of DEPs
In total, 4562 proteins were identified, of which 3648 proteins had quantitative information and annotation terms (Supplementary Table S1). In addition, 505 DEPs were characterized using |fold-change| > 1.3 as the cutoff criterion, of which 299 DEPs were upregulated and 206 DEPs were downregulated.
Subcellular structure classification of DEPs
As shown in Fig. 1, the upregulated DEPs were mainly localized in the cytoplasm (36%), nucleus (25%), extracellular space (20%), mitochondria (7%), and cell membrane (6%) (Fig. 1A); whereas the downregulated DEPs were mainly localized in the extracellular space (28%), cytoplasm (25%), cell membrane (16%), nucleus (12%), and mitochondria (11%).
GO analysis of DEPs
The DEPs were assigned by GOSeq (vision Release2.12) into three groups: biological process (BP), cellular component (CC), and molecular function (MF). As shown in Fig. 2, the upregulated DEPs were mainly enriched in the ribosomes, cytoplasm, cytoplasmic small ribosomal subunits, and ribosomal subunits; whereas the downregulated DEPs were mainly assigned to the extracellular matrix, cytoskeleton, and cell membrane in the CC category. In the MF category, the upregulated DEPs were enriched in the functions of ribosome structure composition, nucleic acid binding, RNA binding, and structural molecular activity; whereas the downregulated DEPs were mainly assigned to Ca2+ binding, extracellular matrix structure composition, protein complex binding, and collagen binding. In the BP category, the upregulated DEPs were mainly assigned to ribosome activation, rRNA processes, endoplasmic reticulum protein synthesis, nuclear transcriptional mRNA catabolism, and other processes; whereas the downregulated DEPs were mainly enriched in extracellular tissues, cellular lipid metabolism, animal organ development, multicellular organisms, and lipid biosynthesis.
KEGG pathway enrichment analysis of DEPs
KEGG analysis was also applied with the Database for Annotation, Visualization, and Integrated Discovery (https://david.ncifcrf.gov/) to functionally enrich gene functions and to identify functional and metabolic pathways. As shown in Fig. 3, the DEPs were mainly enriched in 38 pathways. The upregulated DEPs were mainly enriched in the ribosomal pathway, DNA replication pathway, antigen processing and presentation, and the nucleotide oligomerization domain (NOD)-like receptor pathway, followed by the cell cycle pathway, base repair pathway, interleukin (IL)-17 pathway, drug metabolism pathway, cytokine receptor interaction pathway, malaria, inflammatory bowel disease, and herpes simplex infection (Fig. 3A). The downregulated DEPs were mainly assigned to the fatty acid synthesis pathway, fatty acid metabolism pathway, peroxisome proliferator-activated receptor (PPAR) pathway, glycerophospholipid metabolism pathway, cell adhesion molecule and drug metabolism-P450 pathway, followed by drug metabolism-other enzyme pathway, protein digestion and absorption pathway, cortisol synthesis pathway, pancreatic juice secretion pathway, saliva secretion pathway, and gastric juice secretion pathway (Fig. 3B).
Cluster analysis of DEPs
The DEPs were divided into four groups for cluster analysis of GO and KEGG using the following |fold-change| as the cutoff thresholds: Q1 (0 < Ratio ≤ 1/1.5), Q2 (1/1.5 < Ratio ≤ 1/1.3), Q3 (1.3 < Ratio ≤ 1.5), and Q4 (Ratio > 1.5).
In the BP category, the GO terms of the upregulated DEPs were mainly enriched in protein synthesis, cell differentiation, and activation of inflammatory signaling pathways, such as chronic inflammatory response, T cell activation, NK cell activation, tumor necrosis factor (TNF) production regulation, synthesis of large nucleic acid subunits, cell cycle phase transition, epidermal differentiation, defense response, membrane localization of proteins, and synthesis and decomposition of cellular macromolecules. The downregulated DEPs were mainly related to cell proliferation, including cell responses to carbohydrates and the PPAR signaling pathways, collagen proliferation regulation, lipid metabolism process, cell adhesion molecule regulation, and cytoskeleton development (Fig. 4A).
As shown in Fig. 4B, in the CC category, the upregulated DEPs were mainly related to the body’s cellular immunity such as lysosomes, endoplasmic reticulum membrane space, MHC-II protein complex, nuclear chromosomes, telomere regions, DNA polymerase, and aniline blue particles. The downregulated DEPs were mainly enriched in the extracellular matrix, basement membrane, microfibers, supramolecular polymers, and nucleosomes, which may be closely related to keratinization and proliferation as well as dermal papillary capillary proliferation in the pathogenesis of PV.
In the CC category, the upregulated DEPs were mainly associated with immune defense, including MHC-type II receptor activation, antigen binding, cytokine activation and binding, transferase activity, nucleic acid activation, Toll-like receptor (TLR)-4 binding, helicase activation, and carboxylic acid binding. The downregulated DEPs were mainly related to PV epidermal proliferation such as collagen binding, extracellular matrix binding, and oxidoreductase activity (Fig. 4C).
KEGG cluster analysis revealed that the upregulated pathways included the ribosome pathway, DNA replication pathway, NOD-like receptor pathway, IL-17 pathway, cell circulation pathway, and drug metabolism pathway, which might be associated with protein synthesis, inflammatory activation, and cell proliferation. The downregulation pathways were mainly related to apoptosis, protein recognition, and energy metabolism, including fatty acid biosynthesis pathways, fatty acid biometabolism pathways, PPAR pathways, and glycerophospholipid metabolism pathways (Fig. 4D).
PRM verification
Thirteen target proteins that are involved in two drug metabolism pathways (drug metabolism-other enzymes, drug metabolism-cytochrome P450 pathway) were validated by PRM, of which 10 were quantitated, and 9 of 10 were significant (Table 1). Among them, myeloperoxidase (MPO), thymidine phosphorylase (TYMP), inosine monophosphate dehydrogenase 2 (IMPDH2), glutathione S-transferase mu 4 (GSTM4), and aldehyde dehydrogenase, dimeric NADP-preferring (ALDH3A1) were highly expressed in PV; whereas carboxylesterase 1 (CES1), monoamine oxidase B (MAOB), microsomal glutathione S-transferase 1 (MGST1), and glutathione S-transferase theta-1 (GSTT1) were less expressed in PV tissues (Fig. 5). The three proteins MPO (p/c: 17.05, P < 0.05), TYMP (p/c: 2.44, P < 0.05), and IMPDH2 (p/c: 1.78, P < 0.05) in the drug metabolism-other enzyme pathways were significantly highly expressed in the PV lesional tissues (Fig. 5). The PRM results were consistent with those of TMT-LC-MS/MS, showing a significant increase in the fold change and further confirming the involvement of drug metabolism-other enzyme pathways in the pathogenesis of PV.
Table 1
PRM quantitative results of 9 DEPs
Protein Accession | Protein Gene | con Relative Abundance | pso Relative Abundance | pso/con Ratio | pso/con Ratio (TMT) | pso/con P-value |
P05164 | MPO | 0.11 | 1.89 | 17.05 | 2.04 | 2.92E-09 |
P19971 | TYMP | 0.58 | 1.42 | 2.44 | 1.66 | 7.02E-05 |
Q03013 | GSTM4 | 0.66 | 1.34 | 2.04 | 1.63 | 1.04E-05 |
P12268 | IMPDH2 | 0.72 | 1.28 | 1.78 | 1.35 | 1.25E-04 |
P30838 | ALDH3A1 | 0.80 | 1.20 | 1.50 | 1.41 | 1.56E-05 |
P27338 | MAOB | 1.24 | 0.76 | 0.62 | 0.71 | 9.81E-03 |
P10620 | MGST1 | 1.29 | 0.71 | 0.55 | 0.54 | 1.63E-03 |
P23141 | CES1 | 1.42 | 0.58 | 0.41 | 0.59 | 4.86E-05 |
P30711 | GSTT1 | 1.57 | 0.43 | 0.28 | 0.41 | 3.62E-06 |