Establishment of a Triple Quadrupole HPLC-MS Quantitation Method for Dystrophin Protein in Mouse and Human Skeletal Muscle
Duchenne muscular dystrophy (DMD) is the most common type of neuromuscular disease caused by mutations in the <i>DMD</i> 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...
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MDPI AG
2023-12-01
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| Series: | International Journal of Molecular Sciences |
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| author | Tsukasa Tominari Masaru Takatoya Toshiya Matsubara Michio Matsunobe Daichi Arai Chiho Matsumoto Michiko Hirata Shosei Yoshinouchi Chisato Miyaura Yoshifumi Itoh Hirofumi Komaki Shin’ichi Takeda Yoshitsugu Aoki Masaki Inada |
| author_facet | Tsukasa Tominari Masaru Takatoya Toshiya Matsubara Michio Matsunobe Daichi Arai Chiho Matsumoto Michiko Hirata Shosei Yoshinouchi Chisato Miyaura Yoshifumi Itoh Hirofumi Komaki Shin’ichi Takeda Yoshitsugu Aoki Masaki Inada |
| author_sort | Tsukasa Tominari |
| collection | DOAJ |
| description | Duchenne muscular dystrophy (DMD) is the most common type of neuromuscular disease caused by mutations in the <i>DMD</i> 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. |
| first_indexed | 2024-03-08T15:05:22Z |
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| id | doaj.art-763e91c55e694f5ea2261661ed13a96c |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-08T15:05:22Z |
| publishDate | 2023-12-01 |
| publisher | MDPI AG |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-763e91c55e694f5ea2261661ed13a96c2024-01-10T14:58:55ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-12-0125130310.3390/ijms25010303Establishment of a Triple Quadrupole HPLC-MS Quantitation Method for Dystrophin Protein in Mouse and Human Skeletal MuscleTsukasa Tominari0Masaru Takatoya1Toshiya Matsubara2Michio Matsunobe3Daichi Arai4Chiho Matsumoto5Michiko Hirata6Shosei Yoshinouchi7Chisato Miyaura8Yoshifumi Itoh9Hirofumi Komaki10Shin’ichi Takeda11Yoshitsugu Aoki12Masaki Inada13Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, JapanDepartment of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, JapanLife Science Research Center, Shimadzu Corporation, Nakagyo, Kyoto 604-8511, JapanDepartment of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, JapanDepartment of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, JapanDepartment of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, JapanDepartment of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, JapanCooperative Major of Advanced Health Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, JapanDepartment of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, JapanInada Research Unit, Institute of Global Innovation Research, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, JapanTranslational Medical Center, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8551, JapanDepartment of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8502, JapanDepartment of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8502, JapanDepartment of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, JapanDuchenne muscular dystrophy (DMD) is the most common type of neuromuscular disease caused by mutations in the <i>DMD</i> 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.https://www.mdpi.com/1422-0067/25/1/303dystrophinDuchenne muscular dystrophyLC-MS |
| spellingShingle | Tsukasa Tominari Masaru Takatoya Toshiya Matsubara Michio Matsunobe Daichi Arai Chiho Matsumoto Michiko Hirata Shosei Yoshinouchi Chisato Miyaura Yoshifumi Itoh Hirofumi Komaki Shin’ichi Takeda Yoshitsugu Aoki Masaki Inada Establishment of a Triple Quadrupole HPLC-MS Quantitation Method for Dystrophin Protein in Mouse and Human Skeletal Muscle International Journal of Molecular Sciences dystrophin Duchenne muscular dystrophy LC-MS |
| title | Establishment of a Triple Quadrupole HPLC-MS Quantitation Method for Dystrophin Protein in Mouse and Human Skeletal Muscle |
| title_full | Establishment of a Triple Quadrupole HPLC-MS Quantitation Method for Dystrophin Protein in Mouse and Human Skeletal Muscle |
| title_fullStr | Establishment of a Triple Quadrupole HPLC-MS Quantitation Method for Dystrophin Protein in Mouse and Human Skeletal Muscle |
| title_full_unstemmed | Establishment of a Triple Quadrupole HPLC-MS Quantitation Method for Dystrophin Protein in Mouse and Human Skeletal Muscle |
| title_short | Establishment of a Triple Quadrupole HPLC-MS Quantitation Method for Dystrophin Protein in Mouse and Human Skeletal Muscle |
| title_sort | establishment of a triple quadrupole hplc ms quantitation method for dystrophin protein in mouse and human skeletal muscle |
| topic | dystrophin Duchenne muscular dystrophy LC-MS |
| url | https://www.mdpi.com/1422-0067/25/1/303 |
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