Kazuo Yamada1, Atsushi Watanabe2, Haruo Takeshita3, Ken-Ichi Matsumoto4. 1. Department of Biosignaling and Radioisotope Experiment, Interdisciplinary Center for Science Research, Organization for Research and Academic Information, Shimane University, Enya-cho, Izumo, Shimane 693-8501, Japan; Department of Legal Medicine, Faculty of Medicine, Shimane University, Enya-cho, Izumo, Shimane 693-8501, Japan. 2. Division of Clinical Genetics, Nippon Medical School Hospital, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan; Department of Biochemistry and Molecular Biology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan. 3. Department of Legal Medicine, Faculty of Medicine, Shimane University, Enya-cho, Izumo, Shimane 693-8501, Japan. 4. Department of Biosignaling and Radioisotope Experiment, Interdisciplinary Center for Science Research, Organization for Research and Academic Information, Shimane University, Enya-cho, Izumo, Shimane 693-8501, Japan. Electronic address: matumoto@med.shimane-u.ac.jp.
Abstract
BACKGROUND: Complete deficiency of an extracellular matrix tenascin-X (TNX) leads to a classical type of Ehlers-Danlos syndrome (EDS). TNX haploinsufficiency is a cause of hypermobility type of EDS. Human TNX is also present in a serum form (sTNX) with a molecular size of 140kDa. In this study, we established a method for quantification of sTNX using nano-liquid chromatography tandem mass spectrometry (LC/MS/MS) with selected/multiple reaction monitoring. METHODS: Twelve abundant protein-depleted sera were reduced, alkylated, and digested with Lys-C and trypsin. Subsequently, the digests were fractionated by strong cation exchange chromatography. Optimal and validated transitions of precursor and product ions of the peptides from sTNX were developed on a triple quadrupole mass spectrometer. RESULTS: Serum concentrations of sTNX of healthy individuals were quantified as an average of 144ng/ml. However, sTNX was not detected by this method in serum from a patient with a classical type of EDS in whom sTNX was not found by Western blot analysis. The limit of quantification (LOQ) of sTNX by nano-LC/MS/MS method was 2.8pg whereas the detection sensitivity of sTNX by Western blot analysis was 19pg. The nano-LC/MS/MS method is more sensitive than Western blot analysis. CONCLUSIONS: The quantification method will be useful for diagnosis and risk stratification of EDS caused by TNX deficiency and haploinsufficiency.
BACKGROUND: Complete deficiency of an extracellular matrix tenascin-X (TNX) leads to a classical type of Ehlers-Danlos syndrome (EDS). TNX haploinsufficiency is a cause of hypermobility type of EDS. HumanTNX is also present in a serum form (sTNX) with a molecular size of 140kDa. In this study, we established a method for quantification of sTNX using nano-liquid chromatography tandem mass spectrometry (LC/MS/MS) with selected/multiple reaction monitoring. METHODS: Twelve abundant protein-depleted sera were reduced, alkylated, and digested with Lys-C and trypsin. Subsequently, the digests were fractionated by strong cation exchange chromatography. Optimal and validated transitions of precursor and product ions of the peptides from sTNX were developed on a triple quadrupole mass spectrometer. RESULTS: Serum concentrations of sTNX of healthy individuals were quantified as an average of 144ng/ml. However, sTNX was not detected by this method in serum from a patient with a classical type of EDS in whom sTNX was not found by Western blot analysis. The limit of quantification (LOQ) of sTNX by nano-LC/MS/MS method was 2.8pg whereas the detection sensitivity of sTNX by Western blot analysis was 19pg. The nano-LC/MS/MS method is more sensitive than Western blot analysis. CONCLUSIONS: The quantification method will be useful for diagnosis and risk stratification of EDS caused by TNX deficiency and haploinsufficiency.