In-situ fingerprinting phosphorylated proteins via surface-enhanced Raman spectroscopy: Single-site discrimination of Tau biomarkers in Alzheimer's disease

Protein phosphorylation, a post-translational modification of proteins, is of vital importance in biological regulation. Highly sensitive and site-specific identification of phosphorylated proteins is a key requirement for unraveling crucial signal transduction pathways relevant to cancers and neurodegenerative disorders. Traditional detection methods, however, suffer from relying on antibodies, labels or fragmentation prior to analysis. Here, an antibodyand label-free in situ approach to fingerprint protein phosphorylation was developed based on intrinsic Raman vibrational information of phosphorylated tyrosine, serine, threonine, or histidine residues. Combining surface-enhanced Raman scattering (SERS) spectroscopy and an immobilized-metal affinity strategy, this method is ultrasensitive to discriminate a single-site phosphorylated S396 in a Tau410 protein, an important biomarker in Alzheimer's disease. The binding feasibility of phosphorylated proteins to the modified SERS-active materials is further evidenced by molecular dynamics simulations. This proof-of-concept study paves a new way for the evaluation of site-specific and intact protein phosphorylation in both fundamental mechanical investigation and clinical applications.

Automated summary

The presented method utilizes intrinsic Raman vibrational information of phosphorylated residues to develop an antibody and label-free approach for fingerprinting protein phosphorylation. Through surface-enhanced Raman scattering spectroscopy and an immobilized-metal affinity strategy, this ultrasensitive method can successfully discriminate single-site phosphorylated residues in proteins, demonstrating potential for both fundamental research and clinical applications.