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Mol Cell Proteomics. 2020 Jan 2. pii: mcp.TIR119.001820. doi: 10.1074/mcp.TIR119.001820. [Epub ahead of print]

Concentration determination of >200 proteins in dried blood spots for biomarker discovery and validation.

Author information

1
University of Victoria - Genome BC Proteomics Centre, University of Victoria, Canada.
2
University of Victoria - Genome BC Proteomics Centre, University of Victoria, Victoria, BC, Canada.
3
Department of Pathology and Laboratory Medicine, University of British Columbia, Canada.
4
Island Medical Program, Department of Pathology and Laboratory Medicine, University of British Columbia, Canada.
5
University of Victoria - Genome BC Proteomics Centre.
6
University of Victoria - Genome BC Proteomics Centre, University of Victoria, Victoria, BC, Canada christoph.borchers@mcgill.ca.

Abstract

The use of protein biomarkers as surrogates for clinical endpoints requires extensive multilevel validation including development of robust and sensitive assays for precise measurement of protein concentration.  Multiple reaction monitoring (MRM) is a well-established mass-spectrometric method that can be used for reproducible protein-concentration measurements in biological specimens collected via microsampling.  The dried blood spot (DBS) microsampling technique can be performed non-invasively without the expertise of a phlebotomist, and can enhance analyte stability which facilitate the application of this technique in retrospective studies while providing lower storage and shipping costs, since cold-chain logistics can be eliminated.  Thus, precise, sensitive, and multiplexed methods for measuring protein concentrations in DBSs can be used for de novo biomarker discovery and for biomarker quantification or verification experiments.  To achieve this goal, MRM assays were developed for multiplexed concentration measurement of proteins in DBSs. The lower limit of quantification (LLOQ) was found to have a median total coefficient of variation (CV) of 18% for 245 proteins, while the median LLOQ was 5 fmol of peptide injected on column, and the median inter-day CV over 4 days for measuring endogenous protein concentration was 8%.  The majority (88%) of the assays displayed parallelism, while the peptide standards remained stable throughout the assay workflow and after exposure to multiple freeze-thaw cycles.  For 190 proteins, the measured protein concentrations remained stable in DBS stored at ambient laboratory temperature for up to 2 months.  Finally, the developed assays were used to measure the concentration ranges for 200 proteins in twenty same sex, same race and age matched individuals.

KEYWORDS:

Biofluids*; Biomarker: Diagnostic; Biomarker: Prognostic; Blood*; Dried Blood Spot; HPLC; High Throughput Screening; Mass Spectrometry; Multiple reaction monitoring; Omics; Quantification

PMID:
31896676
DOI:
10.1074/mcp.TIR119.001820
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