Peptide-Level Biomarker as a New Direction for Blood-Based Testing: Evaluation of Plasma Proteome Variability Induced by Prolonged on-Ice Storage

Plasma proteomics has been explored extensively for biomarker discovery for both diagnostic and therapeutic purposes. The gold standard for plasma proteomics sample preparation requires immediate plasma isolation following blood collection, which poses logistical challenges in many clinical settings. To evaluate the feasibility of delayed plasma processing, we investigated the impact of prolonged on-ice storage (i.e., 0/4/8 h postcollection) on plasma proteomic profiles using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow. Each sample was analyzed by a label-free quantitative data-independent MS/MS method against a project-specific spectral library in technical replicates without fractionation, identifying 752 to 1,000 (903 ± 58) protein groups within each sample. While 72–86% of protein groups were qualitatively identified in samples of all three preparation time conditions from the same donor, quantitative analysis revealed significant alterations in the plasma proteome with as little as a 4-h delay in plasma preparation. Notably, von Willebrand factor (VWF), a plasma biomarker currently used in several clinical tests, was found to have decreased levels in samples with delayed processing. Peptide-level analysis revealed that several VWF-derived peptides were less susceptible to degradation during storage. These findings suggest that monitoring stable peptide markers, rather than intact proteins, may provide a more accurate reflection of the in vivo proteomic state and enhance biomarker reliability. Advancing the development of robust, disease-specific peptide panels will be key to realizing the full potential of clinical proteomics for precise and predictive diagnostics.