Antibody-Free Detection of Phosphoserine/Threonine Containing Peptides by Homogeneous Time-Resolved Fluorescence

Protein phosphorylation is a critical signaling mechanism in cellular regulation and stress response, and more than 95% of the phosphorylations are targeted toward Ser or Thr amino-acid residues. The classical techniques for analyzing phospho-amino acid residues use radioisotopes or sequence-specific antibodies. However, both practical and economical limitations have prevented their development, and we here propose an original approach for the detection of phospho-Ser/Thr residues. It requires no antibody and exploits the patented homogeneous time-resolved fluorescence (HTRF) technology, in association with a 3-step chemical transformation of phospho-amino acids into fluorescent derivatives. The process involves: (i) alkaline beta-elimination of the phosphorylated group, (ii) Michael addition of a bifunctional group, and then (iii) introduction of cyanin-5 as fluorescent acceptor for HTRF. The donor fluorescent moiety at the N-terminus of the phosphorylated peptide is a streptavidin europium cryptate conjugate. After its development, the detection system has been validated on synthetic peptide substrates of Chk2, a key protein kinase activated in response to DNA damage and involved in cell cycle arrest. The results showed a good correlation with known specificity profiles. Interestingly, the detection system is versatile, easy to implement, and suitable for multiple parallel analyses.