CRP is generally present in low abundance relative to other serum proteins. As a result, direct quantitation of CRP at normal levels in serum by mass spectrometry is problematic. An affinity purification technique, using a magnetic bead coupled to a monoclonal antibody against CRP, was employed to isolate CRP from the complex mixture of proteins in serum and improve the feasibility of mass spectrometric quantification. A recombinant form of CRP (rCRP) was produced in E. coli with media containing 15N-ammonium chloride as the only nitrogen source. The resulting rCRP was of high purity (> 99%) and possessed a high level of 15N incorporation (> 99%). Mixtures of CRP and 15N-rCRP were combined in specific ratios, subjected to affinity purification, tryptic digestion, and analysis by LC-MS/MS. The results of quantitation were linear over the range of CRP concentrations studied, indicating that CRP and 15N-rCRP exhibit similar antibody binding and digestion characteristics. A commercially available serum control material for CRP was analyzed in a similar manner, using 15N-rCRP as the internal standard, and a value of 1.3 mg/L was obtained, in good agreement with the manufacturer-assigned value. These results indicate that the methodology can be used to quantify CRP in serum at levels of clinical diagnostic significance.
- An affinity purification approach using a monoclonal antibody against CRP has been developed to isolate CRP from human serum.
- An isotope-dilution approach to CRP quantitation has been demonstrated using 15N-rCRP as an internal standard.
- CRP has been quantified in human serum at levels that were previously not achievable by LC-MS/MS.