Proteome-Wide Investigation of Proline Hydroxylation in Pancreatic Ductal Adenocarcinoma Using DiLeu Isobaric Labeling Strategy

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by a dense fibrotic stroma intertwined with a collagen-rich extracellular matrix (ECM), which significantly contributes to tumor progression. In this study, we developed a high-throughput quantitative method that integrates enhanced hydrophilic interaction liquid chromatography (HILIC) with modified elution conditions and 12-plex N,N-dimethyl leucine (DiLeu) isobaric tags, facilitating efficient multiplexed quantitative analysis of hydroxyproline. This approach was applied to human pancreatic samples and resulted in the identification of 194 hydroxyproline peptides from 157 hydroxyproline sites and 59 proline-hydroxylated proteins, representing the first and the largest hydroxyproline proteomics dataset reported for the pancreas to date. This dataset lays a molecular foundation for understanding the structure-function relationships of hydroxyproline-containing proteins and their roles in pancreatic physiology and pathology. We then apply this strategy to investigating proline hydroxylation alterations in benign pancreatic tumors, PDAC, and their normal adjacent tissues (NAT). Our findings suggest significant biological functions related to proline hydroxylation, including altered patterns of key proteins such as collagen alpha-1(I) chain and collagen alpha-1(XII) chain. These proteins emerge as potential targets for further studies on proline hydroxylation in PDAC, potentially elucidating its role in modifying protein structures and influencing cancer progression.