Selective Probing of Acidic Amino Acid Residues for Protein Structural Analysis by Covalent Labeling and Mass Spectrometry

We present a chemical labeling strategy for specific bioconjugation of aspartic (D) and glutamic (E) acid residues, which is crucial for elucidating their roles in protein-protein interactions and potential therapeutic interventions. Traditional methods using carbodiimides are limited by nonspecificity and side-product formation, prompting the development of our reagent, diphenyldiazomethane (DPDAM), which facilitates modification with a single reagent. We demonstrate its efficacy in selectively labeling Asp and Glu at pH 4 and <i>C</i>-terminal specific carboxylic acid modification achievable at lower pH. Notably, this approach offers a streamlined conversion of acids to esters, producing molecular nitrogen as the sole byproduct. Validation through tandem mass spectrometry confirms the fragmentation characteristics of labeled peptide samples. Using model peptides and proteins, including bradykinin, neurotensin, angiotensin I, bovine serum albumin (BSA), and the cat allergen Fel d 1 protein complex, we showcase the versatility and applicability of our method for probing reactive acidic residues in structural biology studies. This innovation opens avenues for precise labeling of Glu and Asp residues, providing invaluable insights into protein structure and function, as well as identifying potential therapeutic targets.