Monitoring plasma protein stability in dried plasma spots
Dried blood spots are currently used in the U.S. to screen newborn babies for a range of metabolic disorders. However, in medical research, blood collection requires long clinical visits, large volumes of blood and is not easily performed on a weekly basis. Dried blood spots can potentially improve the way samples are collected for proteomics analysis because they are easily deployable, less invasive, and inexpensive to store and transport at ambient temperatures, enabling more frequent low volume sample collection in research. To be able to use dried blood spots in proteomics research, it is important to first understand the effect of this method on protein stability over time. In this study, dried plasma spots (DPS) were investigated because plasma is the cell free fluid component of blood that contains circulating proteins, making it an important analytical material for biomedical research. Blood was collected from 20 healthy individuals and pooled before spotting on the filter paper. Six stabilization conditions were investigated over a period of 2 years which comprised 5 DPS conditions and cryopreservation, which is currently the gold standard for biomedical fluid storage. Four of the DPS conditions involved flushing the storage bags with nitrogen: one was on normal filter paper, one had a Ceres nanoparticle filter paper and two had different protease inhibitors added to the plasma before spotting. One method involved storing the dried plasma samples on filter paper under atmospheric conditions. Every three to six months, the samples were digested by trypsin into peptides and analyzed by liquid chromatography mass spectrometry (LC-MS/MS) to identify how peptide identification changed over time. Unique peptides and proteins identified were analyzed for variations in their stability across the period of storage in each method, and their applications in proteomic studies were determined by Gene Ontology analysis. It was found that all conditions had a decrease in peptide and protein identifications over time, with the atmospheric condition seeing the greatest decline. DPS storage samples had fewer peptides and proteins identified at all time point than the cryopreserved samples. Nevertheless, dried plasma samples which contained the lab mixed cocktail of protease inhibitors and those with Ceres nanoparticles had the least variations in the number of peptides and proteins identified over the two years’ period.