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Immunoaffinity Enrichment and Liquid Chromatography-Selected Reaction Monitoring Mass Spectrometry for Quantitation of Carbonic Anhydrase 12 in Cultured Renal Carcinoma Cells.
| Publication Type | Journal Article |
| Authors | Rafalko, A., Iliopoulos O., Fusaro VA, Hancock W., and Hincapie M. |
| Abstract | 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. |
| Year of Publication | 2010 |
| Journal | Analytical chemistry |
| Date Published (YYYY/MM/DD) | 2010/10/11 |
| ISSN Number | 0003-2700 |
| DOI | 10.1021/ac101981t |
| PubMed | http://www.ncbi.nlm.nih.gov/pubmed/20936840?dopt=Abstract |
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