Protein Precipitation to Remove Carbohydrates that Interfere in Protein-Bound Tryptophan Quantification in Soybean Seeds

Tryptophan is one of the nine essential amino acids in humans that can only be obtained through diets and supplements. It is a precursor to many biological processes, such as serotonin, melatonin, kynurenin, and niacin (nicotinamide) vitamin synthesis. The content of tryptophan in foods, such as soybean is an important indicator of nutritional value. Therefore, accurate quantification of tryptophan in soybean is crucial to soybean nutritional improvement. Quantification of soybean protein-bound amino acids first involves acid hydrolysis of total protein to liberate amino acids. However, tryptophan quantification following acid hydrolysis is difficult or impossible due to its reactions with soybean carbohydrates. Therefore, removal of carbohydrates from soy proteins prior to acid hydrolysis is necessary. In this study, we compared four common protein precipitation methods (i.e., methanol, acetonitrile, acetone, and trichloroacetic acid (TCA) protein precipitation methods) to determine the best method to separate soy proteins from carbohydrates, and concluded that acetone provided the highest recovery of soy proteins. Tryptophan content in the precipitated proteins was determined after acid hydrolysis of the proteins using liquid chromatography–tandem mass spectrometry multiple reaction monitoring (LC–MS/MS–MRM). No significant difference in the tryptophan content was found among proteins precipitated with methanol, acetonitrile, and TCA, suggesting that these precipitated proteins have similar compositions. A slightly lower, but statistically significant tryptophan content was found in the acetonitrile-precipitated proteins, suggesting that these proteins contain slightly higher glycosylated proteins.