Selective enrichment of thiophosphorylated polypeptides as a tool for the analysis of protein phosphorylation.
A chemoselective alkylation method is described for the isolation and subsequent identification of thiophosphorylated peptides/proteins. The method involves thiophosphorylation of proteins using adenosine 5'-O-(thiotriphosphate) (ATPgammaS) followed by selective in situ alkylation of the newly thiophosphorylated proteins resulting in a stable covalent bond. The chemoselective alkylation exploits the relatively high nucleophilicity at low pH of the sulfur in thiophosphate residues, whereas the nucleophilicities of phosphates, amines, and other functionality of amino acids are negligible or significantly suppressed. Modified alkylation reagents linked to biotin or solid supports (e.g. glass or Sepharose beads) with or without a photocleavable linker facilitate the isolation of the thiophosphorylated peptide/proteins. This approach is demonstrated through the localization of phosphorylation sites on myosin regulatory light chain. We anticipate that this technique will be useful for isolation and subsequent identification of newly thiophosphorylated proteins, produced either in vivo or in vitro, thus facilitating the dissection of protein phosphorylation networks.
Kwon
Sung Won
SW
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9038, USA.
Kim
Sung Chan
SC
Jaunbergs
Janis
J
Falck
John R
JR
Zhao
Yingming
Y
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D000478:Alkylation; D001710:Biotin / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D018994:Myosin Light Chains / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D010766:Phosphorylation; D011506:Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D018994:Myosin Light Chains; D010455:Peptides; D011506:Proteins; C022571:adenosine 5'-O-(3-thiotriphosphate); D001710:Biotin; D000255:Adenosine Triphosphate