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Analytical chemistry DOI:10.1021/ac101981t

Immunoaffinity Enrichment and Liquid Chromatography-Selected Reaction Monitoring Mass Spectrometry for Quantitation of Carbonic Anhydrase 12 in Cultured Renal Carcinoma Cells.

Publication TypeJournal Article
Year of Publication2010
AuthorsRafalko, A, Iliopoulos, O, Fusaro, VA, Hancock, W, Hincapie, M
JournalAnalytical chemistry
Date Published2010/10/11

Liquid chromatography-selected reaction monitoring (LC-SRM) is a highly specific and sensitive mass spectrometry (MS) technique that is widely being applied to selectively qualify and validate candidate markers within complex biological samples. However, in order for LC-SRM methods to take on these attributes, target-specific optimization of sample processing is required, in order to reduce analyte complexity, prior to LC-SRM. In this study, we have developed a targeted platform consisting of protein immunoaffinity enrichment on magnetic beads and LC-SRM for measuring carbonic anhydrase 12 (CA12) protein in a renal cell carcinoma (RCC) cell line (PRC3), a candidate biomarker for RCC whose expression at the protein level has not been previously reported. Sample processing and LC-SRM assay were optimized for signature peptides selected as surrogate markers of CA12 protein. Using LC-SRM coupled with stable isotope dilution, we achieved limits of quantitation in the low fmol range sufficient for measuring clinically relevant biomarkers with good intra- and interassay accuracy and precision (≤17%). Our results show that using a quantitative immunoaffinity capture approach provides specific, accurate, and robust assays amenable to high-throughput verification of potential biomarkers.