Quantitative, Multiplexed Assays for Low Abundance Proteins in Plasma by Targeted Mass Spectrometry and Stable Isotope Dilution*S

By Hasmik Keshishian, T. Addona, Michael W. Burgess, E. Kuhn, Steven A. Carr. Molecular & Cellular Proteomics, 2007

669 citations recorded by the source corpus.

Biomarker discovery produces lists of candidate markers whose presence and level must be subsequently verified in serum or plasma. Verification represents a paradigm shift from unbiased discovery approaches to targeted, hypothesis-driven methods and relies upon specific, quantitative assays optimized for the selective detection of target proteins. Many protein biomarkers of clinical currency are present at or below the nanogram/milliliter range in plasma and have been inaccessible to date by MS-based methods. Using multiple reaction monitoring coupled with stable isotope dilution mass spectrometry, we describe here the development of quantitative, multiplexed assays for six proteins in plasma that achieve limits of quantitation in the 1–10 ng/ml range with percent coefficients of variation from 3 to 15% without immunoaffinity enrichment of either proteins or peptides. Sample processing methods with sufficient throughput, recovery, and reproducibility to enable robust detection and quantitation of candidate biomarker proteins were developed and optimized by addition of exogenous proteins to immunoaffinity depleted plasma from a healthy donor. Quantitative multiple reaction monitoring assays were designed and optimized for signature peptides derived from the test proteins. Based upon calibration curves using known concentrations of spiked protein in plasma, we determined that each target protein had at least one signature peptide with a limit of quantitation in the 1–10 ng/ml range and linearity typically over 2 orders of magnitude in the measurement range of interest. Limits of detection were frequently in the high picogram/milliliter range. These levels of assay performance represent up to a 1000-fold improvement compared with direct analysis of proteins in plasma by MS and were achieved by simple, robust sample processing involving abundant protein depletion and minimal fractionation by strong cation exchange chromatography at the peptide level prior to LC-multiple reaction monitoring/MS. The methods presented here provide a solid basis for developing quantitative MS-based assays of low level proteins in blood.

Subfields: Molecular Biology, Spectroscopy

View Paper: https://www.semanticscholar.org/paper/74a884cdbe6c4dd624175cdd20f5eb099308112e

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Citations in the corpus

Less label, more free: Approaches in label‐free quantitative mass spectrometry (2011, background): “SRM can determine relative and absolute protein quantities at low attomolar concentrations [115, 116].”
Peptidomic discovery of short open reading frame-encoded peptides in human cells (2012, neutral): “Due to the high charge state of the peptides (+5 ions) the tandem MS (CID) was not informative, which led us to use additional methods for conformation including IDMS of trypsin fragments and cellular imaging experiments.”
Sex estimation using sexually dimorphic amelogenin protein fragments in human enamel (2019, background): “In parallel reaction monitoring the instrumentation focuses exclusively on relevant peptides, increasing the sensitivity of detection, sometimes by orders of magnitude (Keshishian et al., 2007).”