Rapid development of sensitive, high-throughput, quantitative and highly selective mass spectrometric targeted immunoassays for clinically important proteins in human plasma and serum.

OBJECTIVES: The aim of this study was to develop high-throughput, quantitative and highly selective mass spectrometric, targeted immunoassays for clinically important proteins in human plasma or serum. DESIGN AND METHODS: The described method coupled mass spectrometric immunoassay (MSIA), a previously developed technique for immunoenrichment on a monolithic microcolumn activated with an anti-protein antibody and fixed in a pipette tip, to selected reaction monitoring (SRM) detection and accurate quantification of targeted peptides, including clinically relevant sequence or truncated variants. RESULTS: In this report, we demonstrate the rapid development of MSIA-SRM assays for sixteen different target proteins spanning seven different clinically important areas (including neurological, Alzheimer's, cardiovascular, endocrine function, cancer and other diseases) and ranging in concentration from pg/mL to mg/mL. The reported MSIA-SRM assays demonstrated high sensitivity (within published clinical ranges), precision, robustness and high-throughput as well as specific detection of clinically relevant isoforms for many of the target proteins. Most of the assays were tested with bona-fide clinical samples. In addition, positive correlations, (R2 0.67-0.87, depending on the target peptide), were demonstrated for MSIA-SRM assay data with clinical analyzer measurements of parathyroid hormone (PTH) and insulin growth factor 1 (IGF1) in clinical sample cohorts. CONCLUSIONS: We have presented a practical and scalable method for rapid development and deployment of MS-based SRM assays for clinically relevant proteins and measured levels of the target analytes in bona fide clinical samples. The method permits the specific quantification of individual protein isoforms and addresses the difficult problem of protein heterogeneity in clinical proteomics applications.

Model-based discovery of circulating biomarkers.

Proteomic-based biomarker discovery approaches broadly attempt to identify proteins whose basal abundance, or change in abundance in response to a perturbation (e.g., a therapeutic intervention) is able to discriminate between populations of patients. Up until recently, the majority of approaches for discovering circulating biomarkers have focused on directly profiling serum or plasma to identify such proteins. However, the complexity and dynamic range of protein abundance in serum and plasma create a significant challenge for proteomics methods. To overcome these barriers, diverse approaches to simplify or to fractionate serum and plasma have been developed. For some diseases, such as those related to specific organs, there may be useful marker proteins that originate in the organ. Here, we describe an approach for marker discovery that focuses on the profiling of either primary tissue or cell culture models thereof.