Temporal Assessment of Protein Stability in Dried Blood Spots

The use of protein biomarkers in blood for clinical settings is limited by the cost and accessibility of traditional venipuncture sampling. The dried blood spot (DBS) technique offers a less invasive and more accessible alternative. However, protein stability in DBS has not been well evaluated. Herein, we deployed a quantitative LC-MS/MS system to construct proteomic atlases of whole blood, DBSs, plasma, and blood cells. Approximately 4% of detected proteins' abundance was significantly altered during blood drying into blood spots, with overwhelming disturbances in cytoplasmic fraction. We also reported a novel finding suggesting a decrease in the level of membrane/cytoskeletal proteins (SLC4A1, RHAG, DSC1, DSP, and JUP) and an increase in the level of proteins (ATG3, SEC14L4, and NRBP1) related to intracellular trafficking. Furthermore, we identified 19 temporally dynamic proteins in DBS samples stored at room temperature for up to 6 months. There were three declined cytoskeleton-related proteins (RDX, SH3BGRL3, and MYH9) and four elevated proteins (XPO7, RAN, SLC2A1, and SLC29A1) involved in cytoplasmic transport as representatives. The instability was governed predominantly by hydrophilic proteins and enhanced significantly with an increasing storage time. Our analyses provide comprehensive knowledge of both short- and long-term storage stability of DBS proteins, forming the foundation for the widespread use of DBS in clinical proteomics and other analytical applications.