Fecal biomarker discovery by mass spectrometry-based proteomics: a perspective towards non-invasive colorectal cancer screening methods

Colorectal cancer (CRC) represents the second-leading cause of cancer death worldwide. 1 Up to now, screening programs involve the detection of occult blood in stools - characterized by high false-positive rate and, in case of positivity, subsequent colonoscopy, whose invasiveness causes reluctance to perform it. Consequently, non-invasive diagnostic screening tests are strongly required to increase patient engagement, and to improve the prognosis and the efficacy of the pharmacological therapy. Mass spectrometry (MS)- based proteomics could serve as a powerful tool for discovering new disease biomarkers, to further develop reliable diagnostic tests for early and accurate diagnosis. 2,3 Investigation of non-invasive stool samples is crucial to boost population engagement in preventive care screening. Hence, the present work aims to combine the discovery of faecal CRC biomarkers by MS-based proteomics, with the perspective of implementation of immunochemical point-of-care assays based on smart electrochemical sensing devices. Stool samples were collected from patients who underwent colonoscopy and had at least one adenomatous polyp or cancer, stratified according to histology in low-grade dysplasia, high-grade dysplasia, and adenocarcinoma; the study included also healthy controls. Untargeted MS-based shotgun proteomics was performed using a nano-LC coupled to an Orbitrap Exploris 480 with a High-Field Asymmetric Waveform Ion Mobility Spectrometry System. The applied sample preparation procedure involved the use of a lysis buffer combined with sonication and protein precipitation, and permitted to extract an average of 100 proteins per sample. A total of 513 distinct proteins were identified and semi-quantified across all the samples. Statistical analysis highlighted the presence of some enriched human proteins in patients with low-grade dysplasia, high-grade dysplasia and adenocarcinoma with respect to healthy subjects: two most promising CRC biomarkers were shortlisted, here generically named as PROT1 and PROT2 (Figure 1). The upregulation of these proteins throughout cancer progression found confirmation in the literature. The next step of the project will be the development of electrochemical immunosensors for the non-invasive determination of Prot1 and Prot2 in stool samples, through proper optimization of experimental conditions, among which a fast and non-denaturant extraction procedure.