title: Antibody-free detection of phosphoserine/threonine containing peptides by homogeneous time-resolved fluorescence.
abstract: 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 β-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.
authors.affiliation: CEA, DSV, IBEB, SBTN, Bagnols-sur-Cèze F-30207, France. beatrice.alpha-bazin@cea.fr
authors.forename: Béatrice
authors.initials: B
authors.lastname: Alpha-Bazin
authors.forename: Eric
authors.initials: E
authors.lastname: Quéméneur
mesh_terms: D000595:Amino Acid Sequence; D064447:Checkpoint Kinase 2; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D010768:Phosphoserine; D010769:Phosphothreonine; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D010455:Peptides; D010769:Phosphothreonine; D010768:Phosphoserine; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases