Fig 1: The dynamic range of protein fold-changes in comparisons 1 and 3 and the results of upstream regulator analysis in Ingenuity Pathway Analysis (IPA). The dynamic range with marked fold-changes of the 19 final marker candidates in comparisons 1 (LGD versus HGD) (A) and 3 (LGD versus invasive IPMN) (B). The red and blue dots indicate the log2-transformed fold-changes of the 7 upregulated and 12 downregulated proteins. The yellow dot represents the log2-transformed fold-change of CEA protein. (C) Five potential markers (MUC13, CD55, CPS1, SOD2, and LEFTY1) and their upstream regulators are connected by dotted lines. The molecular types of each upstream regulator are shown in parentheses. Each marker candidate is listed in order of decreasing fold-change, whereas the upstream regulators are listed in order of increasing p-values. LGD, low-grade dysplasia; HGD, high-grade dysplasia; INV, invasive IPMN.
Fig 2: Volcano plots of differentially expressed proteins in three comparison groups. Student’s t-test (p < 0.05) was conducted for comparisons 1 (low-grade dysplasia (LGD) versus high-grade dysplasia (HGD)) (A), 2 (HGD versus invasive IPMN) (B), and 3 (LGD versus invasive intraductal papillary mucinous neoplasm (IPMN)) (C) to discover differentially expressed proteins (DEPs). The DEPs that were significantly expressed in each comparison group are indicated as colored dots (red: upregulated DEPs, blue: downregulated DEPs). Several marker candidates, including the validation target CD55, are highlighted in each comparison. DEP, differentially expressed protein.
Fig 3: Validation of CD55 as a potential biomarker target by ELISA. ELISA was performed with CD55-specific antibodies to evaluate the validity of CD55 as a potential biomarker. The concentration patterns of CD55 by ELISA were generally consistent with the LFQ intensities. The CD55 concentrations are indicated according to two types of IPMN classification. The median CD55 concentrations were highest in invasive IPMN (A) and high-risk IPMN (B). LGD, low-grade dysplasia; HGD, high-grade dysplasia; INV, invasive IPMN; MCN, mucinous cystic neoplasm; SCN, serous cystic neoplasm; *, p < 0.05; **, p < 0.01; NS, not available.
Fig 4: Six of the 19 potential markers that were differentially expressed in accordance with the degree of IPMN malignancy. DEFA3 (A), MUC13 (B), CD55 (C), CPS1 (D), AMY2A (E), and CPB1 (F) were differentially expressed, in accordance with the histological grades of IPMN. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; NS, not available.
Fig 5: Experimental workflow. The overall experimental workflow comprises 3 sections: (1) Preparation of 30 individual samples, (2) peptide library construction, and (3) validation by ELISA. The cohort for label-free quantification included 30 pancreatic cyst fluid samples (10 LGD, 5 HGD, 5 invasive IPMN, 5 MCN, and 5 SCN). After mucus removal by sonication, the samples were centrifuged to isolate supernatant. Pooled cyst fluid (comprising equal amounts of 30 individual samples), secreted proteins from PANC1, Mia Paca-2, BxPC3, and pooled cell lysates from the 3 cell lines were compiled to generate a peptide library. All samples were precipitated using cold acetone to extract the protein. After FASP digestion, only the samples that were used to construct the peptide library were subjected to high-pH reverse-phase peptide fractionation. All peptides were analyzed on a Q Exactive mass spectrometer. CD55, one of the potential markers of IPMN dysplasia, was validated by ELISA. PPT, precipitation; FASP, filter-aided sample preparation; LGD, low-grade dysplasia; HGD, high-grade dysplasia; MCN, mucinous cystic neoplasm; SCN, serous cystic neoplasm; SPNT, supernatant.
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