High-Throughput Protein-Bound Amino Acid Quantification from Maize Kernels

In cereal crops, seed quality is determined by the composition and levels of protein-bound amino acids, which account for ∼90% of the seed total amino acid content. In maize particularly, seed quality is affected by the low levels of lysine and tryptophan, two amino acids that humans and animals cannot synthesize and must obtain from the diet. The low levels of these two amino acids in seeds is due to the dominance of seed storage proteins, namely zeins, which are deficient in these two amino acids. Many efforts have been deployed to improve the nutritional composition of maize kernels (i.e., seeds). Still, the lack of high-throughput and inexpensive methods for the quantification of amino acids that are found within proteins has limited those efforts, especially when large populations are targeted. In this protocol, we describe a robust, efficient, and high-throughput method for the quantification of all 20 proteogenic (protein-forming) amino acids from a crude protein extract. The method consists of four major parts: first, release of the 20 proteogenic amino acids from the protein backbone through hydrolysis; second, aqueous extraction of the released amino acids; third, separation, detection, and quantification of the released amino acids using a multiple reaction monitoring-based ultraperformance liquid chromatography-tandem mass spectrometry detection; and fourth, data analysis and processing using the MassLynx data analysis software, TargetLynx.