title: Proteomic analysis of the androgen receptor via MS-compatible purification of biotinylated protein on streptavidin resin.
abstract: The strength of the streptavidin/biotin interaction poses challenges for the recovery of biotinylated molecules from streptavidin resins. As an alternative to high-temperature elution in urea-containing buffers, we show that mono-biotinylated proteins can be released with relatively gentle heating in the presence of biotin and 2% SDS/Rapigest, avoiding protein carbamylation and minimizing streptavidin dissociation. We demonstrate the utility of this mild elution strategy in two studies of the human androgen receptor (AR). In the first, in which formaldehyde cross-linked complexes are analyzed in yeast, a mass spectrometry-based comparison of the AR complex using SILAC reveals an association between the androgen-activated AR and the Hsp90 chaperonin, while Hsp70 chaperonins associate specifically with the unliganded complex. In the second study, the endogenous AR is quantified in the LNCaP cell line by absolute SILAC and MRM-MS showing approximately 127,000 AR copies per cell, substantially more than previously measured using radioligand binding.
authors: Institute for Systems Biology, Seattle, WA 98109, USA.
authors: Ryan J
authors: RJ
authors: Austin
authors: Heidi M
authors: HM
authors: Smidansky
authors: Carly A
authors: CA
authors: Holstein
authors: Deborah K
authors: DK
authors: Chang
authors: Angela
authors: A
authors: Epp
authors: Neil C
authors: NC
authors: Josephson
authors: Daniel B
authors: DB
authors: Martin
mesh_terms: D000595:Amino Acid Sequence; D001710:Biotin / Q000302:isolation & purification* / Q000378:metabolism; D019921:Biotinylation; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000592:standards; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D040901:Proteomics; D011944:Receptors, Androgen / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012015:Reference Standards; D012441:Saccharomyces cerevisiae; D019809:Streptavidin / Q000737:chemistry*
chemical_list: D010446:Peptide Fragments; D011944:Receptors, Androgen; D011993:Recombinant Fusion Proteins; D001710:Biotin; D019809:Streptavidin