Introduction - Colorectal cancer (CRC) is one of the most common cancer types in the world. It predominantly develops from benign polyps and is frequently caused by lifestyle choices. Early detection and treatment of the tumour results in 5-year survival rats of greater 95% however decreases dramatically to about 10% for stage IV. The availability of reliable biomarkers for CRC is sparse. Here we want to use mass spectrometry approach to profile fresh frozen CRC specimen from stage I-IV for early onset biomarker detection. Methods- Seventy human fresh frozen CRC specimen, with at least 10 patients per group, were digested with trypsin and analysed by fixed window DIA on a QExactive mass spectrometer. Pools of stage I-II and stage III-IV were fractionated by basic reversed phase (RP) chromatography for spectral library generation. Additionally, exosome enrichment by centrifugation from serum samples of the same patients (10 per stage) and 10 serum samples from healthy individuals was performed and analysed by data dependent acquisition on a Fusion mass spectrometer. Results - Basic RP chromatography resulted in a spectral library including ~4600 proteins. Data extraction with Skyline revealed 1900 proteins quantifiable across all 70 specimen. Statistical analysis was performed and stage-specific marker proteins were observed. Especially for Stage I compared to the other stages, significant differences were observed. For later stages (III & IV) metastatic markers were significantly increased. For the exosome samples, label free MS1 quantification was performed and ~1400 proteins were quantified. Similar to the tissue specimen, stage-specific protein profiles were observed. With increasing stage, the number of detectable proteins associated to cancerous alteration increased which may be used for blood-based colon cancer diagnosis. Furthermore, exosomes from patients with cancer drastically varied from the healthy individuals. Conclusion - Mass spectrometric characterisation of fresh frozen CRC tissue specimen and serum exosomes identified stage-specific marker proteins with potential application in early cancer onset detection.