A circulating extracellular vesicles‐based novel screening tool for colorectal cancer revealed by shotgun and data‐independent acquisition mass spectrometry

Abstract

2020) A circulating extracellular vesicles-based novel screening tool for colorectal cancer revealed by shotgun and data-independent acquisition mass spectrometry, ABSTRACT Background: Early screening for colorectal cancer (CRC) is essential to improve its prognosis. Liquid biopsies are increasingly being considered for diagnosing cancer due to low invasiveness and high reproducibility. In addition, circulating extracellular vesicles (crEVs, extracellular vesicles isolated from plasma) expressing tumour-specific proteins are potential biomarkers for various cancers. Here, we present a data-independent acquisition (DIA)-mass spectrometry (MS)-based diagnostic method for liquid biopsies. Methods: Extracellular vesicles (EVs) were isolated from culture supernatants of human CRC cell lines, and plasma of patients with CRC at different tumour stages, by overnight ultracentrifugation coupled with sucrose density gradient centrifugation. Tumour-specific EV proteins were prioritized using Tandem Mass Tag (TMT)-based shotgun proteomics and phosphoproteomics. The results were verified in a second independent cohort and a mouse tumour-bearing model using Western blotting (WB). The candidate biomarkers were further validated in a third cohort by DIA-MS. Finally, the DIA-MS methodology was accelerated to permit high-throughput detection of EV biomarkers in another independent cohort of patients with CRC and healthy controls. Results: High levels of total and phosphorylated fibronectin 1 (FN1) in crEVs, haptoglobin (HP), S100A9 and fibrinogen α chain (FGA) were significantly associated with cancer progression. FGA was the most dominant biomarker candidate. Analysis of the human CRC cell lines and the mouse model indicated that FGA+ crEVs were likely released by CRC cells. Furthermore, fast DIA-MS and parallel reaction monitoring (PRM)-MS both confirmed that FGA+ crEVs could distinguish colon adenoma with an area of curve (AUC) in the receiver operating characteristic (ROC) curve of 0.949 and patients with CRC (AUC of ROC is 1.000) from healthy individuals. The performance outperformed conventional tumour biomarkers. The DIA-MS quantification of FGA+ crEVs among three groups agreed with that from PRM-MS. Conclusion: DIA-MS detection of FGA+ crEVs is a potential rapid and non-invasive screening tool to identify early stage CRC.

Discovery and validation of PZP as a novel serum biomarker for screening lung adenocarcinoma in type 2 diabetes mellitus patients

“…SWATH-MS technology can obtain all fragment information of all ions in the sample without omission and difference, while PRM technology can achieve the absolute quantification of protein expression. The combination of the two strategies can be used for the efficient, comprehensive and accurate screening of potential biomarkers [ 29 , 30 ]. In this study, we performed SWATH-MS analysis to identify DEPs specific for LAC in patients with T2DM, and these potential biomarkers were validated by PRM-MS and ELISA analysis in the discovery and validation cohort.…”

Application of exosomes as liquid biopsy in clinical diagnosis

“…Essentially, the most common way applied to decrease contamination is dilution, which is a necessary step before ultracentrifugation. 145 What also caught our attention is that during the preparation of proteins/peptides for MS technology, a solid-phase extraction method will be utilized for lipoprotein removal, 146 which may also be suitable for exosome purification. Minimal Information for Studies of Extracellular Vesicles guidelines (2018) put forward the classification of existing extraction methods based on recovery and specificity of exosomes, 2 which provided a way for researchers to select appropriate and stable methods in subsequent studies.…”

Proteomics of Extracellular Vesicles: Update on Their Composition, Biological Roles and Potential Use as Diagnostic Tools in Atherosclerotic Cardiovascular Diseases

“…MS is an effective approach for identifying potential EV-derived biomarkers or targets for drug treatments in pathological conditions. A DIA-MS-based diagnostic method was recently described to study EV protein and phosphoprotein biomarkers in liquid biopsies of patients with colorectal cancer and the DIA-MS quantifications were then confirmed by a targeted approach known as parallel reaction monitoring-mass spectrometry (PRM-MS) [ 136 ]. A quantitative proteomic analysis based on a stable isotopic labelling method using isobaric tags for relative and absolute quantitation (iTRAQ) of urinary EV proteins was performed to discover potential biomarkers for prostate cancer diagnosis, and then the candidate proteins were verified using multiple reaction monitoring (MRM) MS [ 137 ].…”