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Colorectal cancer (CRC) is the second cause of death in men and the third in women. This work deals with the study of the low molecular weight protein fraction of sera from patients who underwent surgery for CRC and who were followed for several years thereafter. MALDI-TOF MS was used to identify serum peptidome profiles of healthy controls, non-metastatic CRC patients and metastatic CRC patients. A multiple regression model was applied to signals preliminarily selected by SAM analysis to take into account the age and gender differences between the groups. We found that, while a signal m/z 2021.08, corresponding to the C3f fragment of the complement system, appears significantly increased only in serum from metastatic CRC patients, a m/z 1561.72 signal, identified as a prothrombin fragment, has a significantly increased abundance in serum from non-metastatic patients as well. The findings were also validated by a bootstrap resampling procedure. The present results provide the basis for further studies on large cohorts of patients in order to confirm C3f and prothrombin as potential serum biomarkers. Thus, new and non-invasive tests might be developed to improve the classification of colorectal cancer. This work was supported by grants from Italian Ministry of Health (5×1000 Funds 2013 and 2015) and IT8-11/13 from the Basque Government.