LC-ESI-TOF MS Method for the evaluation of the immunostimulating activity of soybeans via the determination of the functional peptide soymetide

This document is the unedited author's version of a Submitted Work that was subsequently accepted for publication in Journal of Agricultural and Food Chemistry, copyright © American Chemical Society after peer review.Bioactive peptides content in foodstuffs can seriously vary with many factors such as crop variety, food processing, animal breeding, etc. Because of this variability, quantitative methodologies are required to evaluate the content of bioactive peptides in foodstuffs. Progress in liquid chromatography and mass spectrometry technologies offer a great opportunity for the quantitation of bioactive peptides. This study undertook the development of a liquid chromatography–electrospray ionization–time-of-flight mass spectrometry method using a fused-core technology column for the sensitive and unambiguous determination of the immunostimulating peptide soymetide in soybean varieties. Soymetide precursor protein (α′ subunit of β-conglycinin) was extracted with a Tris–HCl buffer (pH 8.0) containing urea and digested with trypsin. Soymetide separation conditions by reversed phase liquid chromatography (ion-pairing reagent, organic modifier, separation column, and elution gradient) and detection by MS were optimized, and a study of soymetide stability was also conducted. The method selectivity having been demonstrated, the linearity, accuracy, precision, and limits of detection and quantitation were evaluated. The developed method enabled the detection and quantitation of soymetide concentrations in the ppb range (7.5 ng/mL and 25 ng/mL, respectively), and about 30 times lower than those detected and determined in a previous work by capillary liquid chromatography with UV detection. These values could allow the quantitation of only 17 μg of soymetide per gram of soybean. The developed methodology was applied to the quantitation of soymetide in different soybean varieties from Europe, Japan, and USA observing great differences in soymetide content that ranged from 40 to 600 μg per gram of soybean depending on the soybean variety.Financial support from the Spanish Ministry of Science and Innovation (project CTQ2009-09022), the Comunidad Autónoma of Madrid (Spain) and european funding from FEDER program (project S2009/AGR-1464, ANALISYC-II), and the University of Alcalá and the Comunidad Autónoma of Madrid (project CCG10-UAH/AGR-5950) is gratefully acknowledged. M. Guijarro-Díez thanks the Ministry of Science and Innovation for his predoctoral grant. Thanks to the CRF (Centro de Recursos Fitogenéticos del Instituto Nacional de Investigaciones Agrarias, Madrid, Spain) by donation of soybean varieties