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.
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