Surface enhanced Raman spectroscopy of Aurora kinases: direct, ultrasensitive detection of autophosphorylation

Abstract

Highly sensitive Surface Enhanced Raman Spectroscopy (SERS) was used to study two of the tumorigenic aurora family kinases, Aurora A and Aurora B in sub-picomole quantities. Significantly, the proteins on conjugating to SERS-active citrate-capped silver nanoparticles retain their enzyme activity as demonstrated by kinase assays, making SERS suitable for studies of enzymatic processes. The ability to differentiate between two structurally similar homologous proteins further corroborates the sensitivity of this technique. Based on the available structural information we demonstrate here that SERS could be used for direct and ultra-sensitive detection of autophosphorylation. The overall reduction in SERS intensity and the presence of bands at 952 and ∼1076 cm⁻¹ confirmed the phosphorylated state of Aurora A. Thus, the detection of autophosphorylation in a full length protein is demonstrated for the first time using SERS, which is critical to attain its active conformation.