Temperature- and time-dependent degradation of mouse tissue proteins: insights into RNA-binding protein stability via mass spectrometry†

In proteomics research, samples are frequently stored at −20 °C and −80 °C for extended periods, and assessing protein stability under these conditions is essential. We evaluated protein stability in healthy and diseased mice liver tissues stored at 4 °C, −20 °C, and −80 °C for 0, 7, 30, 90, and 180 days. A 10% variation in protein concentrations (by day 90, p < 0.001) was observed via BCA assay across all conditions. Untargeted proteomic analysis was performed using in-solution trypsin digestion and LC-Q-Orbitrap-MS/MS, with data processed using Proteome Discoverer 2.5. Proteins were shortlisted based on ≥2 unique peptides, FDR < 1%, and abundance ratio p ≤ 0.001. Differentially expressed proteins were identified using log 2 FC ± 2, p-adj ≤ 0.05. Protein degradation varied with storage conditions. In healthy tissues, 24, 11, and 8 proteins completely degraded at 4 °C, −20 °C, and −80 °C, respectively, after 7 days, compared to 8, 2, and 3 proteins in diseased tissues. The total number of significant proteins consistently identified across all time points in healthy samples was 2570, 2711, and 2617, and in diseased samples it was 2124, 2414, and 2353 at 4 °C, −20 °C, and −80 °C, respectively. RNA-binding proteins, such as La ribonucleoprotein 1B, Reticulophagy regulator 3, and Telomerase RNA component interacting RNase, were particularly prone to degradation across all conditions within 7 days. Notably, 18 degraded proteins were reported as biomarkers in disease conditions. Although −20 °C and −80 °C provided better preservation, residual instability persisted. Optimizing storage conditions is essential to prevent degradation, particularly for biomarker discovery studies.