Nanotrap-enabled quantification of KRAS-induced peptide hydroxylation in blood for cancer early detection

Circulating peptide is a potential source of biomarkers for cancer detection. However, the existence of large molecular weight proteins in plasma have a disastrous effect on circulating peptides isolating and detecting. Herein, nanotrap fractionation following by mass spectrometry have been applied to quantify the levels of bradykinin (BK) and hydroxylated bradykinin (Hyp-BK) as a relative measure of KRAS-regulated Prolyl-4-hydroxylase alpha-1 (P4HA1) which may serve as early diagnosis marker for pancreatic ductal adenocarcinoma (PDAC). We found that P4HA1 can be upregulated by KRAS(G12V), which is a PDAC driver mutation, using HPNE/KRAS and HPNE cells. And we revealed that P4HA1 is overexpressed in PDAC tumors, compared to normal and inflamed pancreatic tissues. RNA interference revealed that P4HA1 activity was primarily responsible for Hyp-BK production. Mass spectrometry analysis revealed that plasma Hyp-BK/BK ratio was higher in PDAC than pancreatitis patients and healthy controls, while the area under the receiver operating characteristic (ROC) curve (AUC) is 0.8209 (95%CI, 0.7269-0.9149). The Hyp-BK/BK association with PDAC was reproduced in another cohort, where this ratio was found to increase with advancing tumor stage. These novel findings paved the way for wider applications of Nanotrap coupled mass spectrometry as a powerful tool for revealing biosignatures from plasma.