ABSTRACT: Protein biomarkers in disease conditions offer crucial insights into prognosis and treatment options. In research, where protein samples are frequently stored at -20°C and -80°C for extended periods, assessing protein stability under these conditions is essential. We assessed the stability of proteins in both healthy and diseased mice liver tissues stored at 4°C, -20°C, and -80°C for 0, 7, 30, 90, and 180 days. A significant decrease in protein concentrations (over 10% by day 90, p < 0.001) was observed across all storage conditions in the initial BCA assay Further, we conducted an untargeted proteomics analysis using an in-solution trypsin digestion. Raw data generated via LC-Q-Orbitrap-MS/MS and processed with proteome discoverer 2.5. Significant protein groups were identified based on the presence of ≥ 2 unique peptides, a false discovery rate (FDR) of < 1%, and an abundance ratio p-value of ≤ 0.001. Differentially expressed proteins were filtered by p-value and fold change (log2 FC ±2, p-adj ≤ 0.05). Complete degradation was observed in 24, 11, and 8 proteins in healthy samples, and 8, 2, and 3 proteins in diseased samples at 4°C, -20°C, and -80°C, respectively, after 7 days. Notably, 18 of these unstable proteins are previously reported as key biomarkers in disease conditions. Total number of significant proteins consistently identified across all time points in healthy samples was 2570, 2711, and 2617, and in diseased samples was 2124, 2414, and 2353 at 4°C, -20°C, and -80°C, respectively. While -20°C and -80°C provide better conditions for long-term storage, fluctuations in total protein count highlight some instability across storage durations. Therefore, Immediate analysis is preferable to prevent temperature-related protein degradation.
RAMALINGAM PERAMAN 
