title: Antibody-free peptide substrate screening of serine/threonine kinase (protein kinase A) with a biotinylated detection probe.
abstract: Being different from anti-phosphotyrosine antibodies, anti-phosphoserine- or anti-phosphothreonine-specific antibodies with high affinity for the detection of serine/threonine kinase substrates are not readily available. Therefore, chemical modification methods were developed for the detection of phosphoserine or threonine in the screening of protein kinase substrates based on β-elimination and Michael addition. We have developed a biotin-based detection probe for identification of the phosphorylated serine or threonine residue. A biotin derivative induced a color reaction using alkaline phosphate-conjugated streptavidin that amplified the signal. It was effective for the detection and separation of the target peptide on the resin. The detection probe was successfully used in identifying PKA substrates from peptide libraries on resin beads. The peptide library was prepared as a ladder-type, such that the active peptides on the colored resin beads were readily sequenced with the truncated peptide fragments by MALDI-TOF/MS analysis after releasing the peptides from the resin bead through photolysis.
authors.affiliation: School of Chemical and Biological Engineering, Seoul National University, San 56-1, Shilim-dong, Kwnak-gu, Seoul 151-744, South Korea. redcat00@snu.ac.kr1
authors.forename: Mira
authors.initials: M
authors.lastname: Kim
authors.forename: Yong-Sun
authors.initials: YS
authors.lastname: Park
authors.forename: Dong-Sik
authors.initials: DS
authors.lastname: Shin
authors.forename: Jaehi
authors.initials: J
authors.lastname: Kim
authors.forename: Byung-Gee
authors.initials: BG
authors.lastname: Kim
authors.forename: Yoon-Sik
authors.initials: YS
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology; D001710:Biotin / Q000737:chemistry*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D010769:Phosphothreonine / Q000737:chemistry; D019000:Phosphotyrosine / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D019809:Streptavidin / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000906:Antibodies; D019151:Peptide Library; D010455:Peptides; D010769:Phosphothreonine; D019000:Phosphotyrosine; D001710:Biotin; D019809:Streptavidin; D017868:Cyclic AMP-Dependent Protein Kinases