Label-Free Quantification by Liquid Chromatography-Tandem Mass Spectrometry of the Kunitz Inhibitor of Trypsin KTI3 in Soy Products

The greater awareness of consumers regarding the sustainabilityof food chains has shifted part of the consumption from animal proteinsources to vegetable sources. Among these, of relevance both for humanfood use and for animal feed, is soy. However, its high protein contentis unfortunately accompanied by the presence of antinutritional factors,including Kunitz's trypsin inhibitor (KTI). Now there are fewanalytical methods available for its direct quantification, as theinhibitory activity against trypsin is generically measured, whichhowever can be given by many other molecules and undergo numerousinterferences. Therefore, in this work, a direct label-free liquidchromatography-mass spectrometry (LC-MS) method forthe identification and quantification of trypsin Kunitz inhibitorKTI3 in soybean and derivative products has been developed. The methodis based on the identification and quantification of a marker peptide,specific for the protein of interest. Quantification is achieved withan external calibration curve in the matrix, and the limit of detectionand the limit of quantification of the method are 0.75 and 2.51 mu g/g,respectively. The results of the LC-MS method were also comparedwith trypsin inhibition measured spectrophotometrically, highlightingthe complementarity of these two different pieces of information.

Automated summary

The increasing awareness of consumers on sustainable food chains has caused a shift in consumption from animal protein sources to plant protein sources. Soy, which is both relevant for human food use and animal feed, has a high protein content but is accompanied by antinutritional factors such as Kunitz's trypsin inhibitor (KTI). A direct label-free liquid chromatography-mass spectrometry (LC-MS) method has been developed to identify and quantify Kunitz inhibitor KTI3 in soybean and its derivative products, using a marker peptide specific to the protein of interest. The results of the LC-MS method were compared with spectrophotometric trypsin inhibition, demonstrating the complementarity of these two types of analysis.