A highly-multiplexed MRM-based assay for determination of cardiovascular disease (CVD) status and disease classification has been developed for clinical research. A high-flow system using ultra-high performance LC and an Agilent 6490 triple quadrupole mass spectrometer, equipped with an ion funnel, provided ease of use and increased the robustness of the assay. The assay uses 135 stable isotope-labeled peptide standards for the quantitation of 67 putative biomarkers of CVD in tryptic digests of whole plasma in a 30-min assay. Eighty-five analyses of the same sample showed no loss of sensitivity (<20% CV for 134/135 peptides) and no loss of retention time accuracy (<0.5% CV for all peptides). The maximum linear dynamic range of the MRM assays ranged from 10(3) -10(5) for 106 of the assays. Excellent linear responses (r >0.98) were obtained for 117 of the 135 peptide targets with attomole level limits of quantitation (<20% CV and accuracy 80-120%) for 81 of the 135 peptides. The assay presented in this study is easy to use, robust, sensitive, and has high-throughput capabilities through short analysis time and complete automated sample preparation. It is therefore well suited for CVD biomarker validation and discovery in plasma.

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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 interassay 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. Molecular & Cellular Proteomics 14: 10.1074/mcp.O115.049957, 3094-3104, 2015.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 then spotting a drop onto a collection card. Therefore, DBS sampling is less invasive than venipuncture and does not require a trained phlebotomist. This sampling approach also does not require time-sensitive centrifugation, which is crucial for plasma and serum samples to prevent degradation. 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 also important for sample collection in remote locations that may be without reliable access to a centrifuge and/or a freezer designated for biohazardous materials. Quantitative bioanalytical methods using the DBS methodology have been developed for genomic, metabolomic, and proteomic applications including newborn screening (3, 4), therapeutic drug monitoring (5, 6), toxicology and drugs of abuse (7,8), viral disease management (9, 10), and many others (2, 11).Targeted MS, in particular selected/multiple reaction monitoring (SRM/MRM) using internal standards...