Abstract 4583: Multiplex mass spectrometric immunoassays

Abstract

Abstract Targeted proteomics approaches can facilitate the study of the human protein variations and their association with cancer. In Mass Spectrometric Immunoassays, affinity interactions are utilized to selectively retrieve specific proteins from complex biological fluids, and are followed by mass spectrometric analysis of the targeted proteins. These assays are similar to traditional enzyme immunoassays in that they use antibodies as reagents for affinity retrieval of specific proteins, but instead of enzymatic reaction for indirect protein detection, mass spectrometric analysis is used for direct identification of the proteins and their modifications. This rather straightforward concept is realized through affinity pipette devices that enable high-throughput integration and assaying of thousands of samples per day. Described here is the development of several multiplex mass spectrometric immunoassays toward cancer-related proteins. The project is part of the NCI's Clinical Proteomic Technologies for Cancer initiative to assess and apply proteomic technologies and data resources to solve critical problems in cancer research. Antibodies to several proteins (including one that serves as an internal reference standard - IRS) were immobilized in various ratios in the affinity pipettes, and following analysis of human plasma spiked with constant amount of the IRS, the ratio of the peaks representing the proteins were monitored to determine the optimal ratio of antibodies in the affinity pipettes. Detailed assaying protocols were developed and optimized, including pre-and post-sample rinses, and elution matrix composition. Increasingly-diluted plasma samples were analyzed and a decrease of the protein signals in reference to the IRS signal was observed, indicating non-saturating conditions for the assays. The assays were tested with numerous human plasma samples, and the intra- and inter-assay CVs were determined to be <10%. The results obtained with the MSIA assay were also compared to commercially available ELISA assays. The introduction of mass spectrometric-based assays and platforms into clinical laboratories will facilitate the ongoing biomarker discovery, evaluation, and validation efforts. The multiplex mass spectrometric immunoassays are idealy suited for analysis of protein biomarkers exhibiting post-translational modifications because this protein microheterogeneity can only be detected via mass spectrometry. It is important to note that the assays target intact/native proteins (in contrast to MRM-based peptide assays), and in such are capable of detecting any and all PTMs, without a priori knowledge of their existence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4583.