Identification of a novel phosphorylation site in human androgen receptor by mass spectrometry.
An N-terminal hexahistidine-tagged full-length human androgen receptor protein (His(6)-hAR) was overexpressed and purified to apparent homogeneity in the presence of dihydrotestosterone (DHT) in our previous studies. In-gel trypsin digestion of the purified DHT-bound His(6)-hAR, and tryptic peptide mapping using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS), detected a total of 17 peptides (21% coverage of hAR) with 9 peptides originating from the ligand-binding domain (LBD, 31% coverage of LBD). Amino acid sequencing analysis of the tryptic peptides from a separate in-gel digestion of the His(6)-hAR, using HPLC-coupled electrospray ionization ion trap mass spectrometry (LC/ESI-ITMS and MS/MS), unambiguously confirmed 21 peptides with 19% coverage of the hAR, of which 11 peptides originated from the LBD (35% coverage of LBD). These 21 peptides included 11 out of the 17 peptides detected by MALDI/TOF-MS. In addition, a novel serine phosphorylation site (Ser(308)) within the N-terminal transactivation domain of hAR was identified.
Zhu
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Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, Ohio State University, Columbus, Ohio 43210, USA.
Becklin
R R
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Desiderio
D M
DM
Dalton
J T
JT
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D006639:Histidine / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010455:Peptides / Q000032:analysis; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011944:Receptors, Androgen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry
D010455:Peptides; D011944:Receptors, Androgen; D011993:Recombinant Fusion Proteins; D010768:Phosphoserine; D006639:Histidine; D014357:Trypsin