High-Throughput Free Amino Acid Quantification from Maize Tissues

Amino acids in maize can exist in both a free and protein-bound state. While most amino acids are part of a protein backbone, a small percentage of them remain free and play important biological roles, serving as signaling molecules, nitrogen transporters, osmolytes, and precursors for multiple primary and secondary metabolites. Their levels vary widely especially in maize leaves, depending on the developmental stage and in response to environmental conditions. Therefore, accurate and reliable quantification of free amino acids (FAAs) is vital in any effort aimed at studying their response to developmental and environmental cues. In this protocol, we describe a robust, high-throughput method that quantifies the 20 proteogenic amino acids (i.e., those that can be incorporated into proteins) that are found in the free form in maize tissue. This method consists of three major parts: first, aqueous extraction of FAAs from maize tissue; second, separation, detection, and quantification of all 20 proteogenic amino acids using ultraperformance liquid chromatography-tandem mass spectrometry; and third, data analysis and processing using the MassLynx data analysis software, TargetLynx.