Sensitive and Reproducible LC-MS Quantification of C-Reactive Protein in Plasma: A Potential Biomarker of Inflammation

Significance of the Topic

C-reactive protein (CRP) is a key inflammatory biomarker with low baseline plasma levels in healthy individuals and dramatic elevation in response to tissue injury and chronic disease. Accurate quantification of CRP supports diagnosis and monitoring of rheumatoid arthritis, cardiovascular risk, and cancer-related inflammatory processes. Conventional immunoassays face limitations in specificity, dynamic range, and reproducibility, driving interest in LC–MS as a sensitive and standardized alternative for protein quantification.

Objectives and Study Overview

This study evaluates a rapid, kit-based LC–MS workflow for quantifying endogenous CRP in human and rat plasma. The goals were to reduce digestion time from 24 hours to 2 hours, simplify sample preparation without affinity enrichment, achieve low limits of quantification (LLOQs of 0.025–0.1 µg/mL from 35 µL plasma), and demonstrate reproducible calibration over four orders of magnitude.

Methodology and Instrumentation

Plasma samples (35 µL) spiked with human CRP were digested using the ProteinWorks eXpress Direct Digest Kit (abbreviated 5 min denaturation, 2 h tryptic digestion) followed by mixed-mode µElution SPE cleanup (Oasis MCX) to remove salts, lipids, and excess reagents. Tryptic peptides AFVPFK, ESDTSYVSLK and GYSIFSYATK were selected in silico using Skyline and monitored by MRM.

• Column: ACQUITY UPLC HSS T3, 1.8 µm, 2.1 × 50 mm; 55 °C.
• LC mobile phase: 0.1% formic acid in H2O (A) and acetonitrile (B), gradient 100–10% A over 6.4 min.
• Mass spectrometer: Xevo TQ-XS triple quadrupole, ESI+, optimized cone (35 V) and collision energies for primary and confirmatory transitions.
• Software: MassLynx v4.1 and TargetLynx for data acquisition and quantification.

Main Results and Discussion

The Xevo TQ-XS platform delivered 1.4–3.5× higher signal-to-noise ratios compared to the previous generation, lowering LLOQs to 0.025–0.05 µg/mL. Recoveries for all three peptides exceeded 90% with SPE cleanup. Calibration curves in rat and human plasma were linear over 0.025–100 µg/mL (R2 > 0.997) with 1/x or 1/x2 weighting. QC samples across four plasma lots showed mean accuracies of 89–106% and RSDs < 5%. Endogenous CRP levels in human plasma lots ranged from 0.39 to 18.13 µg/mL, confirmed by primary and secondary MRM transitions.

Benefits and Practical Applications

• Fast turnaround: < 3 h workflow from digestion to quantitation.
• High sensitivity: sub-microgram per milliliter LLOQs in minimal sample volume.
• Broad dynamic range: accurate quantification over four orders of magnitude.
• Improved specificity: mixed-mode SPE reduces matrix interferences.
• Reproducibility: standardized kits and protocols ensure consistent performance.

Future Trends and Opportunities

Advancements in high-throughput protein quantification by LC–MS will emphasize further miniaturization of sample preparation, automated platforms, and integration of isotope-labelled standards. Expanded biomarker multiplexing and standardized workflows may enable broader clinical adoption for inflammatory and disease monitoring.

Conclusion

This kit-based LC–MS method enables sensitive, reproducible, and high-throughput quantification of CRP in plasma without affinity enrichment. It offers a practical alternative to immunoassays, with rapid sample preparation, enhanced dynamic range, and robust performance across diverse plasma matrices.

Reference

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