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...

Saliva is a body fluid with important functions in oral and general health. A consortium of three research groups catalogued the proteins in human saliva collected as the ductal secretions: 1166 identifications-914 in parotid and 917 in submandibular/sublingual saliva-were made. The results showed that a high proportion of proteins that are found in plasma and/or tears are also present in saliva along with unique components. The proteins identified are involved in numerous molecular processes ranging from structural functions to enzymatic/catalytic activities. As expected, the majority mapped to the extracellular and secretory compartments. An immunoblot approach was used to validate the presence in saliva of a subset of the proteins identified by mass spectrometric approaches. These experiments focused on novel constituents and proteins for which the peptide evidence was relatively weak. Ultimately, information derived from the work reported here and related published studies can be used to translate blood-based clinical laboratory tests into a format that utilizes saliva. Additionally, a catalogue of the salivary proteome of healthy individuals allows future analyses of salivary samples from individuals with oral and systemic diseases, with the goal of identifying biomarkers with diagnostic and/or prognostic value for these conditions; another possibility is the discovery of therapeutic targets.