(M1030-06-38) Immunocapture LC-MS/MS Assay for the Biomarker, Troponin Fast (TNNI2) Using a Specific Antibody

Purpose: One of the hallmarks of injured skeletal muscle is the appearance of elevated skeletal muscle proteins in circulation. A biomarker, Troponin Fast (TNNI2) is a surrogate to measure this muscle damage. A specific and sensitive assay for TNNI2 is necessary to understand the muscle damage in severe myopathy patients with Becker and Duchenne muscular dystrophy. Here we report on a specific assay for the LC-MS/MS quantitative analysis of TNNI2 From human plasma.

Methods: For specific immunocapture of biomarker Troponin Fast (TNNI2), a well characterized anti- TNNI2 monoclonal antibody having higher binding affinity and slower dissociation rate with minimal cross reactivity to other Troponin isomers will be used. The immunoaffinity extraction will be performed as follows. Pre-washed monoclonal antibody covalently immobilized tosylactivated magnetic beads will be added to Protein LoBind Eppendorf tubes containing the samples. Followed by a 2-hour incubation step at room temperature, the beads will be washed x 5 times to remove unbound and weaky bound proteins. The bound TNNI2 will be eluted with 0.1% formic acid (FA)/ 50% acetonitrile mix, dried down in a SpeedVac concentrator and proceeded for Trypsin Digestion. Assay description: ICH M10 guidance was used for the development of an LC-MS/MS assay for the measurement of TNNI2 from human plasma. All standard and quality control samples were prepared in a surrogate matrix. Trypsin was used to digest TNNI2 into peptides to be analyzed by LC-MS/MS. Two surrogate peptides (ELEDMNQK and MSADAMLK) were followed with MS/MS transitions 503.7 to 764.3 and 433.7 to 735.5, respectively. A 6500+ Sciex mass spectrometer was used with a 30 series UPLC and autosampler from Shimadzu. A Phenomenex Kinetex C18 (2.1x30 mm, 2.6 µm) column was used to separate the peptides. A flow rate of 0.6 mL/min was used with a gradient mobile phase mixture of acetonitrile/water and 0.1% formic acid.

Results: LC-MS/MS was used to develop a sensitive and selective method for the analysis of TNNI2 from human plasma. The assay had a range from 2-1000 ng/mL with accuracy precision better than 15% across the range. TNNI2 was found to be stable throughout all sample storage and preparation conditions. The assay was found selective including against isoforms of Troponin.

Conclusion: We developed a specific, sensitive, accurate and precise method for the HPLC-MS/MS analysis of TNNI2 from human plasma. This method will be used for upcoming clinical studies to determine the efficacy of therapeutics for the treatment of Duchenne and Becker Muscular Dystrophy.

References: A. Russell, et. al., Muscle Nerve 2021 Jul;64(1):43-49