# Profiling the pancreatic cancer secretome with metabolic glycoengineering

## TL;DR

This study uses a new method to better identify proteins secreted by pancreatic cancer cells, which could help find new biomarkers for detection and monitoring.

## Contribution

A metabolic glycoengineering strategy is introduced to enhance secretome profiling and biomarker discovery in pancreatic cancer.

## Key findings

- MGE-LC–MS/MS identified unique secreted and EV-associated proteins in pancreatic cancer cells.
- ManNAc analogs enhance extracellular vesicle production, improving secretome profiling.
- The method successfully enriched PC-derived glycoproteins from mouse plasma for biomarker discovery.

## Abstract

Profiling the secretome for biomarkers offers an attractive, minimally invasive strategy to detect and monitor cancer. Several challenges, however, must be overcome, including the broad dynamic range of biomolecules in the secretome and the requirement for selective detection of tumor-associated markers. Here, we employed a metabolic glycoengineering (MGE) strategy, using 1,3,4-O-Bu3ManNAz, an azido-tagged, bio-orthogonal metabolic precursor of sialic acid, to label the glycome of pancreatic near-normal and cancer cells to improve conventional LC–MS/MS proteomics–based biomarker discovery. By using this “MGE-LC–MS/MS” approach that incorporates MGE enrichment into conventional LC–MS/MS proteomics, we identified several unique proteins from the secretomes of cancer cells evaluated in vitro. In addition to proteins known to be secreted, we identified several putatively intracellular, non–N-glycosylated proteins, such as β-glucocerebrosidase and paladin, linked to pancreatic cancer (PC) as well as proteins associated with extracellular vesicles (EVs) in PC, such as dCTP pyrophosphatase 1. The identification of EV-associated proteins was consistent with our discovery that ManNAc analogs used in the MGE-LC–MS/MS workflow enhance EV production, creating a more complete secretome profile of PC cells. Pointing toward clinical relevance, we used MGE-LC–MS/MS to enrich PC-derived glycoproteins from plasma harvested from mice bearing xenografted human pancreatic tumors, unambiguously demonstrating that this approach can interrogate the secretomes of cancer cells for biomarker discovery. Finally, we discovered that MGE dramatically improved the production of EVs, which both aids in biomarker discovery (this study) and holds potential to facilitate biomanufacturing of these nascent drugs.