SENSITIVE AND REPRODUCIBLE LC-MS QUANTIFICATION OF C-REACTIVE PROTEIN IN PLASMA FOR CLINICAL RESEARCH

Significance of the Topic

Quantifying C-reactive protein (CRP) in plasma is critical for monitoring inflammation, cardiovascular risk, cancer prognosis, and evaluating therapeutic response in clinical research. Traditional ligand binding assays face limitations in multiplexing, selectivity, and dynamic range. LC-MS/MS methods offer improved specificity, broad quantification windows, and the ability to measure multiple biomarkers concurrently, yet often involve lengthy sample preparation.

Objectives and Study Overview

This study aimed to develop a rapid, sensitive, and reproducible LC-MS/MS workflow for CRP quantification in small plasma volumes. Key goals included reducing sample volume to 35 µL, shortening digestion and cleanup to under 3 hours, and achieving low-nanomolar detection limits with robust precision and accuracy across rat and human plasma matrices.

Methodology

• Sample preparation used a generic kit-based protocol for direct digestion (no reduction/alkylation) of 35 µL plasma via the ProteinWorks eXpress Direct Digestion Kit, completed in 2 hours.
• Peptide cleanup employed the ProteinWorks µElution SPE Clean-Up Kit.
• Three signature tryptic peptides (AFVFPK, ESDTSYVSLK, GYSIFSYATK) were monitored with primary and confirmatory transitions.

Instrumentation

• UPLC system: Waters ACQUITY UPLC with HSS T3 column (1.8 µm, 2.1 × 50 mm) at 0.3 mL/min using a 0.1% formic acid/acetonitrile gradient.
• Mass spectrometer: Waters Xevo TQ-XS triple quadrupole MS in positive electrospray ionization (ESI+) mode, operating in multiple reaction monitoring (MRM).

Main Results and Discussion

• Limit of quantification: 0.025–0.1 µg/mL (1–4 nM) for human CRP peptides.
• Linear dynamic range spanned four orders of magnitude (0.025–100 µg/mL) with R² ≥ 0.997.
• Accuracy across calibration points ranged from 92.9% to 105.1%, and intra- and inter-day precision (RSD) remained below 6%.
• Recovery exceeded 90% for all signature peptides using mixed-mode SPE cleanup.
• Endogenous CRP concentrations in four human plasma lots measured by two peptides agreed within 10% (0.4–18.3 µg/mL).

Benefits and Practical Applications

• Rapid turnaround: total sample preparation and analysis in under 3 hours.
• Low sample requirement: 35 µL of plasma enables studies with limited volume.
• High selectivity and multiplex capability allow simultaneous quantification of additional biomarkers.
• Broad dynamic range supports measurement of both baseline and elevated CRP levels in preclinical and clinical settings.

Future Trends and Possibilities

• Integration with automated robotic platforms to increase throughput.
• Expansion to multiplex panels including cytokines and acute-phase proteins.
• Adoption of microflow or nanoflow LC to further enhance sensitivity.
• Application in precision medicine for individualized inflammation profiling.

Conclusion

A streamlined, kit-based LC-MS/MS workflow has been established for the sensitive and reproducible quantification of CRP in rat and human plasma. The method combines rapid digestion, efficient peptide cleanup, and robust MRM detection to deliver reliable biomarker data from minimal sample volumes.

Reference

• Kun E, Wu J, Karl J, Liao H, Zolg W, Guild B. Quantification of c-reactive protein in the serum of rheumatoid arthritis patients using multiple reaction monitoring mass spectrometry and 13C-labeled peptide standards. Proteomics. 2004; Issue 4:1175–1186.
• Allin KH, Nordestgaard BG. Elevated c-reactive protein in the diagnosis, prognosis, and cause of cancer. Crit Rev Clin Lab Sci. 2011;48(4):155–170.
• Waters Corporation. Sensitive and Reproducible LC-MS Quantification of C-Reactive Protein in Plasma: A Potential Biomarker of Inflammation. Application Note 720005819EN. 2017.