Biotin@DpaZn Molecules Enabled Efficient Enrichment of N‑Phosphopeptides under Neutral Conditions

Protein N-phosphorylation has been garnering increasing
attention
owing to its unique biological functions. The large-scale identification
of protein N-phosphorylation serves as the foundation for exploring
a novel function. Despite the advancements in enrichment methods under
neutral conditions, persistently low enrichment efficiency has long
hindered the progress of this field. In this work, Biotin@DpaZn molecules
were first synthesized for liquid–liquid enrichment, which
enabled the efficient enrichment of N-phosphopeptides under neutral
conditions. This enrichment strategy combined the benefits of low
steric hindrance, high selectivity, and high affinity inherent in
pull-down techniques along with the alkali resistance of agarose microspheres,
which could significantly enhance the enrichment efficiency. Compared
to SiO2@DpaZn, the number of identified N-phosphorylation
sites in E. coli increased from 27
to 58. Moreover, we successfully achieved large-scale N-phosphorylation
identification in Corynebacterium glutamicum under different growth conditions, greatly advancing functional
studies. Overall, we developed a liquid–liquid enrichment method
for protein N-phosphorylation. Our work has expanded the identification
coverage of N-phosphorylation, especially in prokaryotes, facilitating
the exploration of potential functions.