A Trapping-Micro-LC-FAIMS/dCV-MS Strategy for Ultrasensitive and Robust Targeted Quantification of Protein Drugs and Biomarkers

• Published in: Analytical chemistry (Washington) 2024-08, Vol.96 (32), p.13140-13149
• Main Authors: Shen, Qingqing, Pu, Jie, Xue, Chao, Zhang, Ming, Qu, Yang, Huo, Shihan, Belford, Michael, Maxey, Charles, Wijeratne, Neloni, Martins, Claudia, Peterman, Scott, Qian, Wei-Jun, Boeser, Cornelia, Qu, Jun
• Format: Article
• Language: English
• Subjects: Animals; BASIC BIOLOGICAL SCIENCES; Biomarkers; Biomarkers - analysis; Biomarkers - blood; Chromatography, Liquid - methods; Drugs; Humans; Ion Mobility Spectrometry - methods; Ionic mobility; Mass Spectrometry - methods; Medical innovations; Mice; Monoclonal antibodies; Optimization; Parameter robustness; Pharmaceutical Preparations - analysis; Pharmaceutical Preparations - chemistry; Plasma; Protein folding; Proteins; Proteins - analysis; Robustness; Signal to noise ratio; Spectrometry
• ISSN: 0003-2700; 1520-6882; 1520-6882
• DOI: 10.1021/acs.analchem.4c01888

The sensitivity of LC-MS in quantifying target proteins in plasma/tissues is significantly hindered by coeluted matrix interferences. While antibody-based immuno-enrichment effectively reduces interferences, developing and optimizing antibodies are often time-consuming and costly. Here, by leveraging the orthogonal separation capability of Field Asymmetric Ion Mobility Spectrometry (FAIMS), we developed a FAIMS/differential-compensation-voltage (FAIMS/dCV) method for antibody-free, robust, and ultrasensitive quantification of target proteins directly from plasma/tissue digests. By comparing the intensity-CV profiles of the target vs coeluted endogenous interferences, the FAIMS/dCV approach identifies the optimal CV for quantification of each target protein, thus maximizing the signal-to-noise ratio (S/N). Compared to quantification without FAIMS, this technique dramatically reduces endogenous interferences, showing a median improvement of the S/N by 14.8-fold for the quantification of 17 representative protein drugs and biomarkers in plasma or tissues and a 5.2-fold median increase in S/N over conventional FAIMS approach, which uses the peak CV of each target. We also discovered that the established CV parameters remain consistent over months and are matrix-independent, affirming the robustness of the developed FAIMS/dCV method and the transferability of the method across matrices. The developed method was successfully demonstrated in three applications: the quantification of monoclonal antibodies with subng/mL LOQ in plasma, an investigation of the time courses of evolocumab and its target PCSK9 in a preclinical setting, and a clinical investigation of low abundance obesity-related biomarkers. This innovative and easy-to-use method has extensive potential in clinical and pharmaceutical research, particularly where sensitive and high-throughput quantification of protein drugs and biomarkers is required.