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. 2015 Nov;14(11):3094-104.
doi: 10.1074/mcp.O115.049957. Epub 2015 Sep 4.

Multiple Reaction Monitoring Enables Precise Quantification of 97 Proteins in Dried Blood Spots

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Multiple Reaction Monitoring Enables Precise Quantification of 97 Proteins in Dried Blood Spots

Andrew G Chambers et al. Mol Cell Proteomics. 2015 Nov.

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.

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Figures

Fig. 1.
Fig. 1.
Multiplexed Analysis. Extracted ion chromatograms for the 173 peptides (99 proteins) initially quantified by LC/MRM-MS in DBS samples. Natural (NAT) peptides are shown in blue and SIS peptides are shown in red. A total of 4 peptides were later removed during assay refinement (see Results, Final Assay): IPTTFENGR from apolipoprotein D (P05090), AASGTQNNVLR from complement component C7 (P10643), LSSGLVTAALYGR from biotinidase (P43251) and VAVNDAHLLQYNHR from galectin-3 (P17931). Because Apolipoprotein D and galectin-3 also were quantified by other peptides, the final assay consisted of 169 peptides targeting 97 proteins.
Fig. 2.
Fig. 2.
Standard Curves. Representative standard curves for protein quantification. The relative response is plotted as (SIS/NAT) and the dashed lines represent the 95% confidence interval. A, Transthyretin, ALGISPFHEHAEVVFTANDSGPR, endogenous protein concentration 79 μg/ml B, Neutrophil gelatinase-associated lipocalin, ITLYGR, endogenous protein concentration 377 ng/ml).
Fig. 3.
Fig. 3.
Overall Dynamic Range. The determined concentrations for the 99 proteins quantified spanned almost 5 orders of magnitude. Two proteins marked by an asterisk (*) - biotinidase and complement component C7 - were later removed from the assay because of poor precision or stability (see Results, Final Assay). Therefore the final analytical assay quantified a total of 97 proteins.
Fig. 4.
Fig. 4.
Multiple Peptides Per Protein. The agreement of multiple peptides per protein across 6 biological samples. A, The correlation between two peptides from apolipoprotein C-II (P02655). B, Peptide ratio CVs for the 44 proteins quantified by multiple peptides. If >two peptides were quantified for a given protein, then the ratio CV between the highest and lowest measured peptide concentration is shown.
Fig. 5.
Fig. 5.
Precision. The analytical precision for 173 peptide targets for each of the 6 biological samples. A, Intra-assay precision, average 6.2–7.7% CV; B, Inter-assay precision, average 9.7–11.5% CV. Peptides not observed in all replicates for a given biological sample are listed as “NA.”
Fig. 6.
Fig. 6.
Fresh versus Previously Frozen. The difference between fresh and previously frozen whole blood on the measured peptide concentrations in DBS samples. Data shown for biological Sample 1 and data for biological Samples 2–6 are available as supplemental information.
Fig. 7.
Fig. 7.
Stability. The number of stable endogenous peptide targets after storage at various temperatures. Peptides are considered stable if their measured concentration remains within 20% in reference to Day 1.
Fig. 8.
Fig. 8.
DBS versus Whole Blood. The comparison of measured concentrations in DBS and whole blood across 6 biological samples. A, The correlation for lumican (P51884) using SLEDLQLTHNK; B, The DBS : whole blood ratio CVs for peptide measurements.

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