Immunoaffinity enrichment and liquid chromatography-selected reaction monitoring mass spectrometry for quantitation of carbonic anhydrase 12 in cultured renal carcinoma cells.
|
| 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.
|
|---|---|
| Authors | Agnes Rafalko, Othon Iliopoulos, Vincent A Fusaro, William Hancock, Marina Hincapie |
| Journal | Analytical chemistry (Anal Chem) Vol. 82 Issue 21 Pg. 8998-9005 (Nov 01 2010) ISSN: 1520-6882 [Electronic] United States |
| PMID | 20936840 (Publication Type: Evaluation Study, Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't) |
| Chemical References |
|
| Topics |
|
Join CureHunter, for free Research Interface BASIC access!
Realize the full power of the drug-disease research graph!