Integrated amino acid profiling and 4D-DIA proteomics reveal protein quality divergence and metabolic adaptation in Cordyceps species

To explore the differences in protein quality among classic medicinal entomopatho-genic fungi and to evaluate their metabolic adaptability, we analyzed the amino acid composition and proteomic characteristics of Cordyceps sinensis (CS), Cordyceps militaris (CM) and Cordyceps cicadae (CC). Quantitative analysis showed CM contained the highest crude protein and lysine, methionine, threonine, and valine. CS adapted to high-altitude hypoxia and exhibited lower protein but elevated leucine, isoleucine, and histidine contents, which may contribute to membrane stabilization and oxidative stress resistance. CC displayed higher non-essential amino acids such as arginine, pro-line, and tyrosine, reflecting active nitrogen metabolism. Four-dimensional da-ta-independent acquisition (4D-DIA) proteomics identified 495 differentially expressed proteins (DEPs), compared with CS, with CM and CC showing upregulated aspartate aminotransferase 2 (AAT2), carbamoyl-phosphate synthase 1 (CPA1), and arginino-succinate synthase 1 (ASS1) in alanine, aspartate, and glutamate pathways, enhancing amino acid biosynthesis. Branched-chain keto acid dehydrogenase E1 subunit alpha (BCKDHA), and acyl-CoA dehydrogenase short/branched chain (ACADSB) in CM and CC drove the integration of branched-chain amino acid catabolism and TCA cycle, which may explain the observed protein content differences between species. This study pioneers the application of 4D-DIA proteomics in comparing CS, CM, and CC, laying a foundation for elucidating quality divergence mechanisms in medicinal fungi.