Establishment of a Triple Quadrupole HPLC-MS Quantitation Method for Dystrophin Protein in Mouse and Human Skeletal Muscle

Author:

Tominari Tsukasa1ORCID,Takatoya Masaru1,Matsubara Toshiya2,Matsunobe Michio1,Arai Daichi1,Matsumoto Chiho1,Hirata Michiko1,Yoshinouchi Shosei3,Miyaura Chisato1,Itoh Yoshifumi45ORCID,Komaki Hirofumi6,Takeda Shin’ichi7ORCID,Aoki Yoshitsugu7ORCID,Inada Masaki134

Affiliation:

1. Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan

2. Life Science Research Center, Shimadzu Corporation, Nakagyo, Kyoto 604-8511, Japan

3. Cooperative Major of Advanced Health Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan

4. Inada Research Unit, Institute of Global Innovation Research, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan

5. Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford OX3 7FY, UK

6. Translational Medical Center, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8551, Japan

7. Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8502, Japan

Abstract

Duchenne muscular dystrophy (DMD) is the most common type of neuromuscular disease caused by mutations in the DMD gene encoding dystrophin protein. To quantitively assess human dystrophin protein in muscle biopsy samples, it is imperative to consistently detect as low as 0.003% of the dystrophin protein relative to the total muscle protein content. The quantitation of dystrophin protein has traditionally been conducted using semiquantitative immunoblotting or immunohistochemistry; however, there is a growing need to establish a more precise quantitative method by employing liquid chromatography-mass spectrometry (LC-MS) to measure dystrophin protein. In this study, a novel quantification method was established using a mouse experiment platform applied to the clinical quantification of human dystrophin protein. The method using a spike-in approach with a triple quadrupole LC-MS quantitated the amount of dystrophin in wild-type and human DMD transgenic mice but not in DMD-null mice. In conclusion, we established a quantitating method of dystrophin using HPLC-LC-MS with a novel spike-in approach. These results indicate that our methodology could be applied to several LC-MS devices to enable the accurate measurement of dystrophin protein in patients with DMD.

Funder

Japan Society for the Promotion of Science (JSPS) KAKENHI

Publisher

MDPI AG

Subject

Inorganic Chemistry,Organic Chemistry,Physical and Theoretical Chemistry,Computer Science Applications,Spectroscopy,Molecular Biology,General Medicine,Catalysis

Reference31 articles.

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