Identification of a novel phosphorylation site in human androgen receptor by mass spectrometry

Biochem Biophys Res Commun. 2001 Jun 15;284(3):836-44. doi: 10.1006/bbrc.2001.5030.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Cells, Cultured
  • Chromatography, High Pressure Liquid
  • Histidine / genetics
  • Humans
  • Molecular Sequence Data
  • Peptide Mapping
  • Peptides / analysis
  • Phosphorylation
  • Phosphoserine / metabolism
  • Protein Structure, Tertiary
  • Receptors, Androgen / chemistry
  • Receptors, Androgen / genetics
  • Receptors, Androgen / metabolism*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Spectrometry, Mass, Electrospray Ionization
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Trypsin / chemistry

Substances

  • Peptides
  • Receptors, Androgen
  • Recombinant Fusion Proteins
  • Phosphoserine
  • Histidine
  • Trypsin