Expression and characterization of 15N-labeled human C-reactive protein in Escherichia coli and Pichia pastoris for use in isotope-dilution mass spectrometry

Levels of C-reactive protein (CRP) in serum are correlated with inflammation and disease in humans. A higher level quantitative method, such as isotope-dilution mass spectrometry (ID–MS) is needed to compare and standardize the many commercial CRP assays. We compare the expression and purification of 15N-CRP from Escherichia coli and Pichia pastoris and show that the protein isolated from P. pastoris has native pentameric structure along with high isotopic enrichment as shown by software developed specifically for this purpose. When this preparation was mixed in various ratios with unlabeled CRP and tryptic peptides of the mixtures were analyzed by LC–MS/MS, the ratios of heavy and light peaks were tightly correlated with input amounts of each protein. In this report we confirm the suitability of 15N-rCRP as an internal standard in ID–MS. Standardization of CRP assays should help validate the relationship between CRP and human health.

► C-reactive protein (CRP) assays are important for predicting cardiovascular risk.
► Soluble 15N-substituted CRP was purified from Escherichia coli and Pichia pastoris.
► Yield per liter was 50-fold higher in P. pastoris.
► Both proteins were highly substituted with 15N (98%).
► 15N-CRP can be used by mass spectrometry to standardize in vitro diagnostic assays.