Comprehensive Profiling of Free Proteinogenic and Non-Proteinogenic Amino Acids in Common Legumes Using LC-QToF: Targeted and Non-Targeted Approaches

Legumes, a dietary staple for centuries, have seen an influx of conventional and unconventional varieties to cater to human care conscious consumers. These legumes often undergo pretreatments like baking, soaking, or boiling to mitigate the presence of non-proteinogenic amino acids (NPAAs) and reduce associated health risks. The recent tara flour health scare, linked to the NPAA baikiain, emphasizes the need for robust analytical methods to ensure the safety and quality of both traditional and novel plant-based protein alternatives. While traditional techniques provide insights into protein and non-proteinogenic amino acid profiles, modern liquid chromatography-mass spectrometry (LC-MS) offers superior sensitivity and specificity for NPAA detection. This study employed an LC-QToF method with MS/MS analysis to comprehensively map the distribution of free NPAAs and proteinogenic amino acids (PAAs) in various legume samples. A total of 47 NPAAs and 20 PAAs were identified across the legume samples, with at least 7-14 NPAAs detected in each sample. Sulfur-containing NPAAs, such as <i>S</i>-methyl-<i>L</i>-cysteine, γ-glutamyl-<i>S</i>-methyl cysteine, and <i>S</i>-methyl homoglutathione, were predominantly found in <i>Phaseolus</i> and <i>Vigna</i> species. Cysteine and methionine were the sulfur-containing PAAs identified. Gel electrophoresis and soluble protein quantification were also conducted to understand legume protein composition holistically. This orthogonal approach provides a valuable tool for ensuring the overall quality of plant-based proteins and may aid in investigating food poisoning or outbreaks related to such products.