Dried blood spots (DBS) are low-tech solutions to sample gathering, storage and transportation. They can be collected easily by non-medical professionals, do not require strict storage conditions for stability, and are safely transported without needing “hazardous materials” labels. Sample handling and analysis protocols are well established for DBS sampling, and the use of DBS is popular in national screening programs.
Researchers routinely analyze DBS samples by immunoassay, but some are investigating mass spectrometry (MS) methods to increase work flow and ramp up the number of biomarkers measured per assay. In their 2013 paper, Chambers and co-workers build on their previous research to compare tandem MS proteomic analysis of DBS and other dried fluid samples with matched serum, plasma and whole blood in order to establish assay validity and reliable recovery of endogenous proteins.1
Researchers gathered blood samples, with and without potassium-EDTA anticoagulant, from five male and five female healthy, non-fasting individuals. Serum, plasma and whole blood samples were pooled and stored appropriately (frozen for serum and plasma; at 4°C for whole blood) before creating dried fluid samples by spotting 15-µL aliquots onto filter paper cards.
Samples were prepared for analysis by elution from filter paper or extraction of 15-µL fluid samples prior to trypsin digestion. Peptides were separated by reverse-phase chromatography (EASY-nLC II system, Thermo Scientific, Bremen, Germany) coupled with an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific). Data resulting from liquid chromatography–tandem mass spectrometry (LC-MS/MS) were then analyzed using Proteome Discoverer ver. 126.96.36.1999 (Thermo Scientific) and matched against the UniProt–Swiss-Prot (ver. 20110104) Homo sapiens database. Samples were run in triplicate to establish assay coefficients of variation (CVs).
Using an untargeted approach, the researchers show that protein and peptide recovery from dried samples is highly consistent (CVs < 4%). Moreover, results were comparable between dried spots and their matched fluid samples, with similar numbers of proteins and peptides identified in each format. There was also an 88%, 83% and 70% overlap in proteins identified in fluid samples versus their dried spot equivalents for serum, plasma and whole blood, respectively.
Interestingly, compared with plasma and serum (fluid and dried spot), whole blood peptide counts were lower, whereas whole blood protein recovery was higher. The authors consider that this is possibly due to erythrocyte lysis during trypsin digestion. Indeed, the presence of the erythrocyte proteome could also explain the higher number of proteins identified exclusively in whole blood samples as compared with serum and plasma. Researchers found only 30–33% overlap in proteins identified in whole blood versus plasma or serum samples.
Altogether, results do demonstrate the validity of LC-MS/MS for analysis of dried fluid samples, along with the potential for multi-analyte assay of this format in the future.
1. Chambers, A.G., et al. (2013) “Comparison of proteins in whole blood and dried blood spot samples by LC/MS/MS,” J. Am. Soc. Mass Spectrom., DOI: 10.1007/s13361-013-0678-x.
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