期刊
ANALYTICAL BIOCHEMISTRY
卷 413, 期 1, 页码 30-35出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2011.02.005
关键词
Ser/Thr kinase; Combinatorial chemistry; Peptide library; Phospho-peptide; beta-Elimination
资金
- Korea Biotech R&D Group of the Next-Generation Growth Engine Project [F104AB010005-07A0201-00510]
- Seoul RBD program [10538]
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 beta-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. (C) 2011 Elsevier Inc. All rights reserved.
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