Quantitative Mass Spectrometric Multiple Reaction Monitoring Assays for Major Plasma Proteins*

By Leigh Anderson, C. Hunter. Molecular & Cellular Proteomics, 2006

1,214 citations recorded by the source corpus.

Quantitative LC-MS/MS assays were designed for tryptic peptides representing 53 high and medium abundance proteins in human plasma using a multiplexed multiple reaction monitoring (MRM) approach. Of these, 47 produced acceptable quantitative data, demonstrating within-run coefficients of variation (CVs) (n = 10) of 2–22% (78% of assays had CV <10%). A number of peptides gave CVs in the range 2–7% in five experiments (10 replicate runs each) continuously measuring 137 MRMs, demonstrating the precision achievable in complex digests. Depletion of six high abundance proteins by immunosubtraction significantly improved CVs compared with whole plasma, but analytes could be detected in both sample types. Replicate digest and depletion/digest runs yielded correlation coefficients (R2) of 0.995 and 0.989, respectively. Absolute analyte specificity for each peptide was demonstrated using MRM-triggered MS/MS scans. Reliable detection of L-selectin (measured at 0.67 μg/ml) indicates that proteins down to the μg/ml level can be quantitated in plasma with minimal sample preparation, yielding a dynamic range of 4.5 orders of magnitude in a single experiment. Peptide MRM measurements in plasma digests thus provide a rapid and specific assay platform for biomarker validation, one that can be extended to lower abundance proteins by enrichment of specific target peptides (stable isotope standards and capture by anti-peptide antibodies (SISCAPA)).

Subfields: Molecular Biology, Spectroscopy

View Paper: https://www.semanticscholar.org/paper/8a0b5b78053d204274dd5bc9c024df7dc21d0c3c

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Citations in the corpus

Less label, more free: Approaches in label‐free quantitative mass spectrometry (2011, methodology): “SRM has been widely employed for over 30 years in small molecule analysis as a tool in the pharmaceutical and analytical chemistry [109] and has been routinely used as a highthroughput and reproducible method of measuring metabolites, hormones, and pesticides in complex sample backgrounds [110–113].”
Protein biomarker discovery and validation: the long and uncertain path to clinical utility (2006, methodology): “have also been measured in plasma or serum using this approach, and Anderson and Hunte”
Full dynamic range proteome analysis of S. cerevisiae by targeted proteomics (2009, methodology): “To alleviate these limitations, we demonstrate in this study the potential of selected reaction monitoring (SRM)-based targeted MS (Anderson and Hunter, 2006; Lange et al., 2008) to provide specific assays for the detection and quantification of proteins over the whole range of cellular concentratio”
The Fundamental Flaws of Immunoassays and Potential Solutions Using Tandem Mass Spectrometry (2009, methodology): “…utilizing MRM approaches can simultaneously quantify dozens of analytes in one specimen, making mass spectrometry an exciting method for not only solving the problems inherent to immunoassays, but for multiplexing protein measurements in the clinical laboratory as well (Anderson and Hunter, 2006).”
Quantitative, Multiplexed Assays for Low Abundance Proteins in Plasma by Targeted Mass Spectrometry and Stable Isotope Dilution*S (2007, background): “concatenated peptide sequences (22, 27)) can be made but requires considerable resources for expression, purification, and characterization of the final protein product.”
The Skyline Ecosystem: Informatics for Quantitative Mass Spectrometry Proteomics (2017, background): “Other Skyline Peptide Settings that affect results of peptide list generation are common biochemical sample preparation concerns such as missed cleavages, oxidized methionine, and peptide amino acid length (Anderson and Hunter 2005; Lange et al. 2008; Prakash et al. 2009).”
Systematic discovery of in vivo phosphorylation networks. (2007, background): “…for 53BP1 phosporylation in vivo. mode termed multiple reaction monitoring (MRM) is well suited to quantify the extent to which a given site is phosphorylated, by measuring speciﬁc fragment ions related to selected peptides (Anderson and Hunter, 2006; Ong and Mann, 2005; Schmelzle and White, 2006).”
The Human Proteome Project: Current State and Future Direction (2011, background): “The short-term goal (within 2 years) is the completion of libraries of such peptides for at least one product of each gene, with critical knowledge of transitions in mass spectra based on fragment ion spectra derived from these peptides using different types of mass spectrometers (4).”
PeptideAtlas: a resource for target selection for emerging targeted proteomics workflows (2008, background): “…be gained on the suitability of individual peptides and transitions. indeed, several recent papers have published lists of validated transitions (anderson & Hunter, 2006; lange et al , 2008). to facilitate the reusability of validated transitions, we have implemented a peptide annotation system…”