Abstract
The dried blood spot (DBS) methodology provides a minimally invasive approach to sample collection and enables room-temperature storage for most analytes. DBS samples have successfully been analyzed by liquid chromatography multiple reaction monitoring mass spectrometry (LC/MRM-MS) to quantify a large range of small molecule biomarkers and drugs; however, this strategy has only recently been explored for MS-based proteomics applications. Here we report the development of a highly multiplexed MRM assay to quantify endogenous proteins in human DBS samples. This assay uses matching stable isotope-labeled standard peptides for precise, relative quantification, and standard curves to characterize the analytical performance. A total of 169 peptides, corresponding to 97 proteins, were quantified in the final assay with an average linear dynamic range of 207-fold and an average R(2) value of 0.987. The total range of this assay spanned almost 5 orders of magnitude from serum albumin (P02768) at 18.0 mg/ml down to cholinesterase (P06276) at 190 ng/ml. The average intra-assay and inter-assay precision for 6 biological samples ranged from 6.1-7.5% CV and 9.5-11.0% CV, respectively. The majority of peptide targets were stable after 154 days at storage temperatures from -20 °C to 37 °C. Furthermore, protein concentration ratios between matching DBS and whole blood samples were largely constant (<20% CV) across six biological samples. This assay represents the highest multiplexing yet achieved for targeted protein quantification in DBS samples and is suitable for biomedical research applications.
Highlights
The dried blood spot (DBS)1 methodology provides several advantages over traditional plasma or serum samples throughout the entire pre-analytical workflow including sample collection, transportation, and storage [1, 2] These blood samples are typically generated using a small sterile lancet to prick the skin and spotting a drop onto a collection card
Li et al demonstrated that capillary devices from two manufacturers (Drummond and Safe-Tec) were both capable of precisely generating DBS samples using only 5 l of blood (Ͻ5% coefficient of variation (CV)) [43]
It is possible that the use of a transfer capillary may be difficult for inexperienced patients at home but this method should certainly be feasible for professionally trained staff, such as a general nurse in a remote clinic location [41, 42]
Summary
The dried blood spot (DBS) methodology provides several advantages over traditional plasma or serum samples throughout the entire pre-analytical workflow including sample collection, transportation, and storage [1, 2] These blood samples are typically generated using a small sterile lancet to prick the skin and spotting a drop onto a collection card. Many analytes have been determined to be stable in the DBS format at room temperature, eliminating the cost associated with cold-chain logistics for sample transportation and storage. These considerations are important for sample collection in remote locations that may be without reliable access to a centrifuge and/or a freezer designated for biohazardous materials. The integration of DBS sampling with MRM is well-established for quantifying a wide range of small molecules (16 –18) This is the standard analytical approach for population-wide screening of newborns for errors in metabolism by targeting amino acids, fatty acid acylcarnitines, and organic acid acylcarnitines [3, 4]. We have developed a multiplexed LC/MRM-MS assay to quantify 97 proteins in DBS samples that is suitable for biomedical research applications
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
