A High-Throughput Absolute Quantification of Protein-Bound Tryptophan from Model and Crop Seeds

This protocol describes a high-throughput absolute quantification protocol for the aromatic essential amino acid, tryptophan (Trp). This procedure consists of a milligram-scale alkaline hydrolysis followed by an absolute quantification step using a multiple reaction monitoring tandem mass spectrometric (LC-MS/MS) detection method. The approach facilitates the analysis of a few hundred samples per week by using a 96-well plate extraction setup. Importantly, the method uses only ∼4 mg of tissue per sample and uses the common alkaline hydrolysis protocol, followed by water extraction that includes L-Trp-d5 as an internal standard to enable the quantification of the absolute level of the bound Trp with high precision, accuracy, and reproducibility. The protocol described herein has been optimized for seed samples for Arabidopsis thaliana, Glycine max, and Zea mays but could be applied to other plant tissues. © 2023 Wiley Periodicals LLC. Basic Protocol: Analysis of protein-bound tryptophan from seeds.