A method for quantification of serum tenascin-X by nano-LC/MS/MS

• Published in: Clinica chimica acta 2016-08, Vol.459, p.94-100
• Main Authors: Yamada, Kazuo, Watanabe, Atsushi, Takeshita, Haruo, Matsumoto, Ken-ichi
• Format: Article
• Language: English
• Subjects: Adult; Chromatography, Liquid; Ehlers-Danlos syndrome; Ehlers-Danlos Syndrome - blood; Humans; Male; Nano-LC/MS/MS; Nanotechnology; SRM/MRM; Stable isotope-labeled peptide; Tandem Mass Spectrometry; Tenascin - blood; Tenascin - deficiency; Tenascin-X
• ISSN: 0009-8981; 1873-3492
• DOI: 10.1016/j.cca.2016.05.022

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. 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. 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. The quantification method will be useful for diagnosis and risk stratification of EDS caused by TNX deficiency and haploinsufficiency. •A method was developed for quantification of sTNX using nano-LC/MS/MS.•Excellent linear regressions were achieved with LOQ in nanogram per milliliter range.•This method will be useful for diagnosis and risk stratification of EDS caused by TNX deficiency and haploinsufficiency.