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Structural analysis of the thyroid hormone receptor ligand binding domain: studies using a quadrupole time‐of‐flight tandem mass spectrometer
Author(s) -
Jonsson Andreas P.,
Carlquist Mats,
Husman Bolette,
Ljunggren Jan,
Jörnvall Hans,
Bergman Tomas,
Griffiths William J.
Publication year - 1999
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/(sici)1097-0231(19990930)13:18<1782::aid-rcm714>3.0.co;2-w
Subject(s) - chemistry , tandem mass spectrometry , mass spectrometry , protein mass spectrometry , tandem mass tag , peptide sequence , chromatography , electrospray , isobaric labeling , mass spectrum , thyroid hormone receptor , analytical chemistry (journal) , receptor , proteomics , biochemistry , quantitative proteomics , gene
The overall architecture of the ligand binding domain (LBD) of members of the nuclear receptor superfamily are similar. There are now standard procedures to express and purify these proteins. A rapid and sensitive method for the structural analysis of these proteins is nano‐electrospray tandem mass spectrometry. In the present study we have analysed the LBD of the human thyroid hormone receptor‐β‐1 (TR‐β) by quadrupole time‐of‐flight tandem mass spectrometry. The intact protein was analysed in a carboxymethylated form in an attempt to identify which cysteine residues are located on the surface. The protein molecular weight (31 652.5 Da) was determined with an accuracy of ±1 Da, while masses of tryptic fragments were determined with an accuracy of at least 75 ppm. The sequence coverage of the tryptic peptide mass map was 93.2 %. Tryptic peptides were subjected to collision‐induced dissociation (CID) and the resulting product ions were mass measured with an accuracy of about 100 ppm. When accurate mass measurements were made with internal calibration, mass accuracies were improved to ±2 ppm in mass spectra, and ±20 ppm in CID spectra. From these data it was possible to determine the presence of post‐translational modifications, locate the sites of carboxymethylation and, in addition, confirm the amino acid sequence of the expressed protein. To the best of our knowledge, this is the first characterisation of the TR‐LBD‐β at the protein level. Copyright © 1999 John Wiley & Sons, Ltd.