There is a move away from the conventional plasma proteomics workflow where highly fractionated samples are analysed using long LC-MS runtimes. Although such workflows can identify many thousands of proteins, their use for large scale discovery proteomics is impractical due to difficulties in scalability. Recent advances involve a shift to bead-based enrichment methods, which enable more proteins to be identified by decreasing the dynamic range.
Our approach to blood proteomics has been increasingly focused on dried blood spots using small volume (30 µL) volumetric absorptive microsampling (VAMS) devices. This has several advantages: the convenience of sample collection (small volume and no special equipment or personnel required); the ease of storage and transport; the accessibility of at-home and remote community sampling; and the application of these to longitudinal studies. A potentially significant downside of dried blood spots is the very wide dynamic range, predominantly driven by the presence of haemoglobin.
We have developed a series of methods that enable the VAMS dried blood spot devices to operate as sample preparation tools as well as collection vessels. Dried whole blood samples are fractionated via sequential extraction with various wash solutions and in-tip digests. These methods have been found to successfully decrease the dynamic range of dried whole blood and enhance detection of the blood proteome. Using DIA-MS we quantify greater than 3,500 proteins from dried whole blood, 2,500 from single spun plasma and 1,200 from double spun plasma. These methods are highly reproducible with CVs between 10-15%.
To further evaluate the utility of VAMS devices we compared sample preparation of whole blood, single and double spun plasma using a commercially available bead-based sample enrichment kit. For both methods, whole blood produced more than 3000 protein IDs, but the bead-based assay introduced significantly more variation compared to VAMS (CV >20% and <10% respectively). Although double-spun plasma produced a lower %CV for the bead method, only 1000 IDs were achieved.
Overall, we have demonstrated the VAMS devices work as both sample collection and preparation devices. Their binding characteristics produce results for single and double spun plasma that are comparable to commercially available products. In addition, VAMS devices provide significant advantages for preparing whole blood.