Mass spectrometric quantitation of C-reactive protein using labeled tryptic peptides

Given the extensive efforts applied toward proteomics and research in biomarkers, methods for the simultaneous measurement of proteins, peptides, metabolic intermediates, hormones, etc. in a complex sample may be required in the foreseeable future. Assays based on mass spectrometric detection may be suitable for meeting the demands of such complex samples with sensitivity and specificity. An analytical method for the quantitation of C-reactive protein (CRP), a well-known marker of inflammation, is described. Exact quantities of two synthetic 13C-labeled CRP tryptic peptides were added as internal standards directly to the sample prior to chemical treatment, trypsinization, and liquid chromatography/mass spectrometry quantitation. C-reactive protein levels based on isotopic response ratios were measured. Intact C-reactive protein was spiked into blank rat urine for chemical and enzymatic treatment, producing linear response ratios of labeled to unlabeled peptides. For rigorous quantitation, standard curves, and quality control samples were prepared in rat urine with highly purified labeled and unlabeled peptides over the 50 pg–5 ng/μL concentration range. Using the same chemical and enzymatic treatment used for digestion of intact CRP, data from these samples demonstrated excellent analytical performance. The method was successfully applied toward the quantitation of urinary C-reactive protein from a study of drug-induced nephrotoxicity.