Multi-site assessment of the precision and reproducibility of multiple reaction monitoring–based measurements of proteins in plasma

Abstract

Verification of candidate biomarkers relies upon specific, quantitative assays optimized for selective detection of target proteins, and is increasingly viewed as a critical step in the discovery pipeline that bridges unbiased biomarker discovery to preclinical validation. Although individual laboratories have demonstrated that multiple reaction monitoring (MRM) coupled with isotope dilution mass spectrometry can quantify candidate protein biomarkers in plasma, reproducibility and transferability of these assays between laboratories have not been demonstrated. We describe a multilaboratory study to assess reproducibility, recovery, linear dynamic range and limits of detection and quantification of multiplexed, MRM-based assays, conducted by NCI-CPTAC. Using common materials and standardized protocols, we demonstrate that these assays can be highly reproducible within and across laboratories and instrument platforms, and are sensitive to low µg/ml protein concentrations in unfractionated plasma. We provide data and benchmarks against which individual laboratories can compare their performance and evaluate new technologies for biomarker verification in plasma.Proteomic technologies based on mass spectrometry (MS) have emerged as preferred components of a strategy for discovery of diagnostic, prognostic and therapeutic protein biomarkers. Because of the stochastic sampling of proteomes in unbiased analyses and the associated high false-discovery rate, tens to hundreds of potential biomarkers are often reported in discovery studies. Those few that will ultimately show sufficient sensitivity and specificity for a given medical condition must thus be culled from lengthy lists of candidates -a particularly challenging aspect of the biomarker-development pipeline and currently its main limiting step. In this context, it is highly desirable to verify, by more targeted quantitative methods, the levels of candidate biomarkers in body fluids, cells, tissues or organs from healthy individuals and affected patients in large enough sample numbers to confirm statistically relevant differences 1, 2. Verification of novel biomarkers has relied primarily on the use of sensitive, specific, high-throughput immunoassays, whose development depends critically on the availability of suitable well-characterized antibodies. However, antibody reagents of sufficient specificity and sensitivity to assay novel protein biomarkers in plasma are generally not available. The high cost and long development time required to generate high-quality immunoassay reagents, as well as technical limitations in multiplexing immunoassays for panels of biomarkers, is strong motivation to develop more straightforward quantitative approaches exploiting the sensitivity and molecular specificity of mass spectrometry.Recently, multiple reaction monitoring (MRM) coupled with stable isotope dilution (SID)-MS for direct quantification of proteins in cell lysates as well as human plasma and serum has been shown to have considerable promise 3- RESULTS Study de...

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

“…SRM mass spectrometry in combination with stable isotope dilution is a powerful approach for protein quantification and offers major advantages over antibody‐based strategies (Addona et al , 2009). Not only does the method circumvent the need for antibody production, but it also provides information about absolute rather than relative amounts of protein.…”

“…Comprehensive studies on the proteomes of cells, tissues, or organisms have been reported (e.g. Addona et al , 2009; Nilsson et al , 2010; Beck et al , 2011; Nagaraj et al , 2011; for review, see Bensimon et al , 2012; Kulak et al , 2014; Wilhelm et al , 2014; Hein et al , 2015; Richards et al , 2015), but accurate quantitative information on proteins expressed at low levels remains scarce. As a consequence, published estimates for the abundance of specific proteins sometimes vary over several orders of magnitude.…”

Antibody-based detection of protein phosphorylation status to track the efficacy of novel therapies using nanogram protein quantities from stem cells and cell lines

“…Numerous approaches can be taken to characterize protein state in biological samples which either employ targeted mass spectrometry (MS) or indirect analysis using antibody-based techniques. MS methods such as selected reaction monitoring (SRM) 28 , also known as multiple reaction monitoring (MRM), are highly sensitive capable of quantitatively detecting attomolar concentrations of protein in complex biological samples (for review see Hjelle 29 ). Antibodybased techniques, which can detect down to femtomolar protein concentrations, include western blot, ELISA, reverse-phase protein arrays (RPPA) and flow cytometry as well as the cIEF assay described here.…”

Applications of targeted proteomics in systems biology and translational medicine

“…This program was renewed in 2011 as the CPTAC, which began focusing on applications 77. CPTAC member laboratories applied standardized methods of multiple reaction monitoring (MRM) and demonstrated reproducibility, precision, sensitive quantitation in tissues and biofluids 78. Cox and coworkers performed a similar interlaboratory precision study of IGF‐1 in plasma across 130 healthy human samples and 22 samples from patients with acromegaly, finding excellent reproducibility 79.…”