An Undesirable Side Effect of EDC-catalyzed Carboxyl Labeling Approach

ABSTRACT: Carbodiimide catalyzed carboxyl and amine conjugation (amidation) has been widely used to protect carboxyl groups. N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (EDC) is the most commonly used carbodiimide reagent in protein chemis-try due to its high efficiency and high solubility in aqueous media. It has also been applied in different proteomics studies including protein terminomics, protein glycosylation and crosslinking. Herein, we report that the EDC catalyzed amidation could cause a +155 Da side-modification at the tyrosine residue and severely hamper the identification of Tyr-containing peptides. We discovered the extremely low identification rate of Tyr-containing peptides in different published studies that employed EDC-catalyzed amidation. We carried out a systematic study and discovered a +155 Da side-modification occur-ring specifically on Tyr as the addition of EDC. Consideration of the side-modification in database search enabled the identification of 13 times more Tyr-containing peptides. Furthermore, we demonstrated that the treatment with carbonate-containing buffer could effectively reduce the side-product. Our study also implies that chemical reactions in proteomic studies should be carefully evaluated prior to their wide applications.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Whole Body

SUBMITTER: Qingqing Li  

LAB HEAD: Xumin Zhang

PROVIDER: PXD020042 | Pride | 2020-12-10

REPOSITORIES: Pride