Phosphoproteomic analysis with a solid-phase capture-release-tag approach.
A comprehensive study of global phosphorylation events in biological systems is critical. We report a chemistry-based capture-release-tag method for isolation of complex phospho-Ser/Thr-containing peptides by liquid beta-elimination combined with solid-phase Michael addition. The free thiol groups of 6-(mercapto-acetylamino)-hexanoic acid functionalized resin are used as immobilized Michael donors to capture dehydro-serine/threonine peptides. After an acid-mediated release step, phospho-peptides are labeled with a 6-(2-mercapto-acetylamine)-hexanoic amide tag at phosphorylated sites. We applied this method to analyze the phosphorylation status of microtubule-associated proteins. We find that a CDK5 substrate microtubule-associated protein 2 (MAP2) is phosphorylated on residues that are within a homologous region of Tau. The chemical method corroborates previous results and suggests that Tau and MAP2 may contain a CDK5 phosphorylation motif.
Tseng
Huang-Chun
HC
Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02155, USA. huang-chun_tseng@hms.harvard.edu
Ovaa
Huib
H
Wei
Nancy J C
NJ
Ploegh
Hidde
H
Tsai
Li-Huei
LH
D000595:Amino Acid Sequence; D001665:Binding Sites; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D003543:Cysteamine / Q000737:chemistry; D007553:Isotope Labeling / Q000379:methods*; D008869:Microtubule-Associated Proteins / Q000032:analysis / Q000737:chemistry; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000032:analysis* / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis / Q000737:chemistry; D010769:Phosphothreonine / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D016875:tau Proteins / Q000737:chemistry / Q000378:metabolism
D008869:Microtubule-Associated Proteins; D010750:Phosphoproteins; D016875:tau Proteins; D010769:Phosphothreonine; D010768:Phosphoserine; D003543:Cysteamine; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases