Colorectal cancer (CRC) is one of the most common cancer types worldwide. It predominantly develops from benign polyps and is frequently caused by lifestyle choices. Early detection and treatment results in 5-year survival rats of greater 95% however decreases dramatically to about 10% for stage IV. Availability of reliable biomarkers for CRC is sparse. Here we use mass spectrometry to profile stage I-IV CRC specimen for early onset biomarker detection.
Fresh frozen CRC tissue specimen, with at least 10 patients per group (71 in total), were analyzed by fixed window Data Independent Acquisition (DIA) Mass spectrometry 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, extracellular vesicle (EV) enrichment from serum samples of CRC patients and serum samples from healthy individuals were performed and analyzed by label-free data dependent acquisition on a Fusion Tribrid mass spectrometer.
Basic RP chromatography resulted in a spectral library including about 4600 proteins. Data extraction with Skyline revealed about 1900 proteins quantifiable across all 71 specimens. Statistical analysis was performed which identified stage-specific marker proteins. Especially for Stage I compared to the other stages, significant differences were observed. For later stages (III & IV) malignancy markers were significantly increased.
For the EV samples, label-free MS1 quantification was performed which quantified ~1400 proteins. The EV content from CRC patients drastically varied from the healthy individuals. With increasing stage, the number of detectable proteins associated to cancerous alteration increased. However, even the EV content from stage I CRC patients demonstrated distinct differences compared to healthy controls.
Quantitative proteome analysis of tissue and extracellular vesicle revealed potential markers for early CRC onset detection which may be used for blood-based colon cancer diagnosis.