title: A method for quantification of serum tenascin-X by nano-LC/MS/MS.
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.
authors.affiliation: 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.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Yamada
authors.affiliation: 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.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Watanabe
authors.affiliation: Department of Legal Medicine, Faculty of Medicine, Shimane University, Enya-cho, Izumo, Shimane 693-8501, Japan.
authors.forename: Haruo
authors.initials: H
authors.lastname: Takeshita
authors.affiliation: 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.
authors.forename: Ken-Ichi
authors.initials: KI
authors.lastname: Matsumoto
mesh_terms: D000328:Adult; D002853:Chromatography, Liquid; D004535:Ehlers-Danlos Syndrome / Q000097:blood*; D006801:Humans; D008297:Male; D036103:Nanotechnology; D053719:Tandem Mass Spectrometry; D019063:Tenascin / Q000097:blood* / Q000172:deficiency
chemical_list: D019063:Tenascin; C081865:tenascin X
keywords: Ehlers-Danlos syndrome; Nano-LC/MS/MS; SRM/MRM; Stable isotope-labeled peptide; Tenascin-X