C-reactive protein (CRP) is a serum analyte of clinical interest in ranges which are difficult to detect using standard mass spectrometry approaches. This paper describes a measurement procedure utilizing affinity purification of intact CRP prior to tryptic digestion and liquid chromatography- tandem mass spectrometry (LC-MS/MS) in order to develop a higher order reference measurement procedure for the certification of reference materials in appropriate ranges. The absence of a suitable internal standard for the CRP measurement, necessary to eliminate potential measurement bias in both the affinity purification and trypsin digestion steps, was addressed using the method of standard addition. The standard addition quantification approach was combined with affinity purification, using an anti-CRP monoclonal antibody conjugated to polystyrene beads, trypsin digestion of the purified protein, and isotope dilution LC-MS/MS analysis of CRP tryptic peptides. This methodology was evaluated through the measurement of CRP in several serum-based CRP control materials, yielding levels that were comparable to their expected mean concentration values. Quantitative results were confirmed with an external calibration approach. This study demonstrates the feasibility of coupling affinity purification with LC-MS/MS for the quantification of low abundance protein analytes in serum with higher order reference measurement procedures necessary for generation of clinical reference materials.
Citation: Analytical Chemistry
Pub Type: Journals
CRP, LC-MS/MS, mass spectrometry, monoclonal antibody, Affinity MS