Impact of single freeze-thaw cycles on human serum proteins: Implications for mass spectrometry biomarker validation

Validation of biomarkers for clinical diagnostics requires high-quality samples and standardized protocols. This study investigated the effect of a single freeze-and-thaw (FT) cycle on the quality of liquid nitrogen-stored clinical serum samples. Using mass spectrometry, serum protein abundance was analyzed in a human test cohort of 99 samples and a validation cohort 109 samples. After preprocessing, 213 proteins were analyzed in the test cohort and 248 in the validation cohort. Thirty proteins changed significantly in their measurability after freeze-thaw in the test cohort, and 11 were confirmed in the validation cohort. Among them, CP and IGHV6-1 showed potential for distinguishing malignant from benign pancreatic disease. No association was found between protein intensity and storage duration. Overall, the findings show that freeze-thaw cycles are an important pre-analytical factor that should be controlled when translating protein biomarkers into clinical practice.