Quantitative proteomic analysis of pancreatic cyst fluid proteins associated with malignancy in intraductal papillary mucinous neoplasms

Abstract

Background: The application of advanced imaging technologies for identifying pancreatic cysts has become widespread. However, accurately differentiating between low-grade dysplasia (LGD), high-grade dysplasia (HGD), and invasive intraductal papillary mucinous neoplasms (IPMNs) remains a diagnostic challenge with current biomarkers, necessitating the development of novel biomarkers that can distinguish IPMN malignancy.

Methods: Cyst fluid samples were collected from nine IPMN patients (3 LGD, 3 HGD, and 3 invasive IPMN) during their pancreatectomies. An integrated proteomics approach that combines filter-aided sample preparation, stage tip-based high-pH fractionation, and high-resolution MS was applied to acquire in-depth proteomic data of pancreatic cyst fluid and discover marker candidates for IPMN malignancy. Biological processes of differentially expressed proteins that are related to pancreatic cysts and aggressive malignancy were analyzed using bioinformatics tools such as gene ontology analysis and Ingenuity pathway analysis. In order to confirm the validity of the marker candidates, 19 cyst fluid samples were analyzed by western blot.

Results: A dataset of 2992 proteins was constructed from pancreatic cyst fluid samples. A subsequent analysis found 2963 identified proteins in individual samples, 2837 of which were quantifiable. Differentially expressed proteins between histological grades of IPMN were associated with pancreatic diseases and malignancy according to ingenuity pathway analysis. Eighteen biomarker candidates that were differentially expressed across IPMN histological grades were discovered-7 DEPs that were upregulated and 11 that were downregulated in more malignant grades. HOOK1 and PTPN6 were validated by western blot in an independent cohort, the results of which were consistent with our proteomic data.

Conclusions: This study demonstrates that novel biomarker candidates for IPMN malignancy can be discovered through proteomic analysis of pancreatic cyst fluid.

Keywords: Biomarker candidates; IPMN dysplasia; Intraductal papillary mucinous neoplasm (IPMN); LC–MS/MS; Pancreatic cyst fluid.

Fig. 1

Detailed experimental workflow. Pancreatic cyst fluid samples from nine individuals (3 LGD, 3 HGD, and 3 invasive IPMN) were included in this study. After centrifugation, pellets and debris were discarded, and the supernatant was collected for proteomic analysis. Proteins in each individual sample and pooled cyst fluid (comprised of equal portions of individual samples) were precipitated with cold acetone. Following FASP digestion, the pooled cyst fluids were fractionated using two types of high-pH fractionation methods. Every prepared sample was analyzed on a Q Exactive mass spectrometer

Fig. 2

Overlap of identified proteins in the individual samples and peptide library and comparison of identified and quantified proteins. a All identified proteins in the nine individual samples and peptide library; 91.9% of proteins were identified both in the individual samples and peptide library. b All identified proteins and quantified proteins in the nine individual samples; 95.7% of quantifiable proteins overlapped with the identified proteins

Fig. 3

Identified and quantified proteins in three individual samples for each histological grade. Identified proteins of three biological replicates in LGD (a), HGD (b), and invasive IPMN (c). Quantified proteins of three biological replicates in LGD (d), HGD (e), and invasive IPMN (f) (INV: invasive IPMN)

Fig. 4

Dynamic range of quantified proteins. Distribution of expression intensities of quantified proteins show a large dynamic range of abundance, but 95% of the proteins were expressed within four orders of magnitude. Several tumor marker proteins, such as MUC2, CEA, and KRAS, were quantified

Fig. 5

Pearson correlation coefficients of technical replicates (TRs) and biological replicates in each histological group. a–c Pearson correlation coefficients of technical replicates in each histological group. Three types of marks indicate the Pearson correlation coefficients of each comparison between technical replicates (◆TR1 vs. TR2, ■TR1 vs. TR3, ▲TR2 vs. TR3). Red dots represent the average Pearson correlation coefficient in each of the 3 comparisons. a–c indicate LGD, HGD, and invasive IPMN, respectively. Box plot representation of Pearson correlation coefficients between each biological replicate in LGD (d), HGD (e), and invasive IPMN (f)

Fig. 6

Volcano plots based on p values in all comparison groups. To determine the differentially expressed proteins, Student’s t test was performed with a Benjamini–Hochberg FDR value of 0.05. The colored dots indicate the proteins that passed the t test for significance between LGD versus HGD (a), HGD versus invasive IPMN (b), and LGD versus invasive IPMN (c). The blue dots represent downregulated proteins, and the red dots denote upregulated proteins

Fig. 7

Venn diagram of differentially expressed proteins in three comparison groups. By Student’s t test (Benjamini–Hochberg FDR = 0.05), 149, 48, and 98 proteins were differentially expressed between LGD and HGD, between HGD and invasive IPMN, and between LGD and invasive IPMN, respectively. A total 243 proteins were found to be DEPs when overlapping components of the Venn diagram were excluded; 49 proteins were shared in at least two comparison groups and are highlighted in white

Fig. 8

Six biomarker candidates among 18 proteins that had expression patterns that were consistent with the degree of IPMN malignancy. HOOK1 (a), PTPN6 (b), and MUC2 (c) were predominantly expressed in invasive IPMN. FBN1 (d), CLDN18 (e), and SERPINA5 (f) were primarily expressed in LGD (*<p value 0.05; **<p value 0.01, ***<p value 0.001, ****<p value 0.0001, NS not significant)

Fig. 9

Validation of HOOK1 and PTPN6 as potential biomarker targets by western blot. A total of 19 pancreatic cyst fluid samples were analyzed by western blot to validate the relative abundance of HOOK1 and PTPN6. The immunoblotting results were consistent with our MS results. a HOOK1 was overexpressed in high-risk IPMN in the proteomic (p value < 0.001) and western blot analyses (p value < 0.01). b PTPN6 was overexpressed in high-risk IPMN in the proteomic (p value < 0.001) and western blot analyses (p value < 0.05) (*<p value 0.05, **<p value 0.01, ***<p value 0.001, ****<p value 0.0001, NS not significant, INV invasive IPMN)