Tyrosine–EDC Conjugation, an Undesirable Side Effect of the EDC-Catalyzed Carboxyl Labeling Approach

Carbodiimide-catalyzed carboxyl and amine conjugation (amidation) has been widely used to protect carboxyl groups. N-(3-(Dimethylamino)propyl)-N′-ethylcarbodiimide (EDC) is the most common carbodiimide reagent in protein chemistry due to its high catalytic efficiency in aqueous media. The reaction has also been applied in different proteomic studies including protein terminomics, glycosylation, and interaction. Herein, we report that the EDC-catalyzed amidation could cause a +155 Da side modification on the tyrosine residue and severely hamper the identification of Tyr-containing peptides. We revealed the extremely low identification rate of Tyr-containing peptides in different published studies employing the EDC-catalyzed amidation. We discovered a +155 Da side modification occurring specifically on Tyr and decoded it as the addition of EDC. Consideration of the side modification in a database search enabled the identification of 13 times more Tyr-containing peptides. Furthermore, we successfully developed an efficient method to remove the side modification. Our results also imply that chemical reactions in proteomic studies should be carefully evaluated prior to their wide applications. Data are available via ProteomeXchange with identifier PXD020042.