Strategies for Monitoring Serum Protein Degradation With an Antibody Array-Based Technology

Human serum is an ideal body fluid for discovering and monitoring biomarkers for disease diagnosis and treatment response. However, intrinsic proteolytic degradation during sample handling may compromise biomarker integrity, which may affect the accuracy of results. To address this issue, we aimed to test the feasibility of using antibody array technology to evaluate the temporal stability of serum proteins at room temperature.

Concentrations of 480 serological proteins were monitored using antibody arrays in samples from 10 healthy donors incubated at 25°C for 0, 6, 12, 24, and 48 h. Linear regression assessed time-dependent concentration changes. Physicochemical properties (molecular weight, isoelectric point, instability index, aliphatic index, hydropathicity) of the proteins were analyzed. Enrichment analyses were performed on degraded proteins.

During 48-h incubation, 201 proteins showed a significant negative linear correlation between concentration and time, among which the concentration of 91 proteins reduced over 20% in the first 6 h. Degraded proteins were significantly associated with lower molecular weight (MW < 40 kDa) but no other physicochemical properties. Enrichment analyses revealed degraded proteins associated with various terms and involved in important signaling pathways, like JAK-STAT, PI3K-Akt, and MAPK.

Our data demonstrate the feasibility of employing antibody arrays for detecting serum protein degradation. Using this platform, we found some serum proteins with clinical significance rapidly degrade at room temperature, including interleukins (e.g., IL-1β/IL-12p40/IL-17), growth factors (e.g., aFGF, bFGF, and BMP-2), and chemokines (e.g., I-309, 6Ckine, and BLC). These may serve as potential biomarkers for assessing human serum sample quality.