Abstract
Dried blood spot (DBS) sampling, coupled with multiple reaction monitoring mass spectrometry (MRM-MS), is a well-established approach for quantifying a wide range of small molecule biomarkers and drugs. This sampling procedure is simpler and less-invasive than those required for traditional plasma or serum samples enabling collection by minimally trained personnel. Many analytes are stable in the DBS format without refrigeration, which reduces the cost and logistical challenges of sample collection in remote locations. These advantages make DBS sample collection desirable for advancing personalized medicine through population-wide biomarker screening. Here we expand this technology by demonstrating the first multiplexed method for the quantitation of endogenous proteins in DBS samples. A panel of 60 abundant proteins in human blood was targeted by monitoring proteotypic tryptic peptides and their stable isotope-labeled analogs by MRM. Linear calibration curves were obtained for 40 of the 65 peptide targets demonstrating multiple proteins can be quantitatively extracted from DBS collection cards. The method was also highly reproducible with a coefficient of variation of <15% for all 40 peptides. Overall, this assay quantified 37 proteins spanning a range of more than four orders of magnitude in concentration within a single 25 min LC/MRM-MS analysis. The protein abundances of the 33 proteins quantified in matching DBS and whole blood samples showed an excellent correlation, with a slope of 0.96 and an R(2) value of 0.97. Furthermore, the measured concentrations for 80% of the proteins were stable for at least 10 days when stored at -20 °C, 4 °C and 37 °C. This work represents an important first step in evaluating the integration of DBS sampling with highly-multiplexed MRM for quantitation of endogenous proteins.
Highlights
Dried Blood Spot (DBS)1 samples have many advantages over blood serum or plasma and are the preferred clinical sample for newborn screening for metabolic diseases [1, 2]
Hemoglobin is the only protein that is commonly targeted in DBS samples, and primary screening is accomplished by high-performance liquid chromatography (HPLC) or isoelectric focusing (IEF) methods [2]
They analyzed more than 2000 DBS samples by targeting tryptic peptides that were unique to four different beta-globin mutations and compared their results with a standard IEF method that measured the intact proteins obtained from corresponding whole blood samples
Summary
Multiplexed Quantitation of Endogenous Proteins in Dried Blood Spots by Multiple Reaction Monitoring - Mass Spectrometry□S. The addition of stable isotope-labeled internal standards (SIS) enables the acquisition of highly reproducible results across a variety of instrumentation at different institutions It is common for 20 –30 small molecule targets including amino acids, fatty acid acylcarnitines, and organic acid acylcarnitines to be analyzed by flow injection MRM-MS, at a cost of $10 –20 USD per patient sample [11]. Boemer et al used a similar flow injection MRM-MS strategy to screen newborns for hemoglobin variants associated with sickle cell disease [15] They analyzed more than 2000 DBS samples by targeting tryptic peptides that were unique to four different beta-globin mutations and compared their results with a standard IEF method that measured the intact proteins obtained from corresponding whole blood samples. The quantitative results from whole blood and the corresponding DBS samples were compared, and the integrity of DBS samples stored under various temperatures has been evaluated
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