Multiple Reaction Monitoring Enables Precise Quantification of 97 Proteins in Dried Blood Spots.
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.
From the ‡University of Victoria - Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101 - 4464 Markham St., Victoria, BC V8Z 7X8, Canada;
Andrew G
AG
Chambers
From the ‡University of Victoria - Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101 - 4464 Markham St., Victoria, BC V8Z 7X8, Canada;
Andrew J
AJ
Percy
From the ‡University of Victoria - Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101 - 4464 Markham St., Victoria, BC V8Z 7X8, Canada;
Juncong
J
Yang
From the ‡University of Victoria - Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101 - 4464 Markham St., Victoria, BC V8Z 7X8, Canada; §Department of Biochemistry and Microbiology, University of Victoria, Petch Building Room 207, 3800 Finnerty Rd., Victoria, BC V8P 5C2, Canada christoph@proteincentre.com.
Christoph H
CH
Borchers
D001681:Biological Assay; D001798:Blood Proteins / Q000302:isolation & purification*; D002853:Chromatography, Liquid; D059788:Dried Blood Spot Testing / Q000295:instrumentation / Q000379:methods / Q000592:standards*; D006801:Humans; D007553:Isotope Labeling; D010455:Peptides / Q000302:isolation & purification*; D055550:Protein Stability; D040901:Proteomics; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry
D001798:Blood Proteins; D010455:Peptides