**4. Conclusions**

The development of non-invasive modalities with high patient compliance that can unequivocally detect and diagnose early stage CRC will afford the greatest opportunity for early intervention strategies to treat asymptomatic patients and ultimately improve the survival of patients with CRC. However, current screening methods are inadequate and there remains a pressing need to establish reliable biomarkers of early stage CRC disease. The resolution that is now achievable with advanced quantitative MS-based proteomic workflows and instrumentation hold the promise of unlocking the secrets of early stage disease that could be exploited to prevent or cure CRC. However, the inherent issues that have plagued MS-based biomarker discovery projects over the last 20 years moderate optimism. Sample size, particularly in the early stage setting, coupled with the wide dynamic range of blood plasma and the observed low concentrations of early stage specific individual protein biomarkers and the lack of reproducibility of MS investigations have meant that no new biomarkers of early stage CRC have entered the clinical setting since the discovery of CEA.

Over the coming years, the limitations of most current MS-based biomarker discovery projects will be resolved, mostly thanks to the recent developments in sophisticated techniques and technologies that not only simplify pre-analytical issues but address analytical limitations. Improvements in sample preparation techniques that potentially do away with immunodepletion, or the enrichment techniques that are currently absolutely necessary for the successful implementation of MS-based plasma biomarker investigation, will increase the reproducibility of future projects [127]. Analytic techniques that employ wider MS/MS windows for the simultaneous detection and quantification of low-abundant potential biomarkers using SWATH-MS strategies are important developments that will continue to arm our ever-evolving arsenal of MS technologies and resolve the issue of both detection and quantitation of low-abundant potential marker of early stage disease.

It is likely in the age of proteogenomics, that the greatest increase in resolution of early stage disease markers will come from the high-throughput simultaneous detection and quantification of protein and non-protein based biomarkers. Indeed, the combination of ctDNA and protein biomarkers in patient plasma with resectable pancreatic ductal adenocarcinomas showed a staggering 99.5% specificity, providing hope of early stage diagnosis for one of the most aggressive forms of gastrointestinal cancer [128]. Non-invasive blood tests combining non-protein and protein biomarkers represents an exciting approach for the early detection of any cancer type and holds the greatest potential for the increased survival of CRC patients worldwide.
