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 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 crosslinked 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.

Improved mass spectrometry-based activity assay reveals oxidative and metabolic stress as sirtuin-1 regulators

“…1A and B ), but the recovery rate of biotinylated peptide remained below 10%. Competition with free biotin [ [53] , [54] , [55] ] in Tris buffer followed by desalting of the peptide with C18 Zip-tips resulted in a 50% higher recovery rate, so we used this protocol in all subsequent experiments ( Supplemental Fig. 2 A and B).…”

The WNT target SP5 negatively regulates WNT transcriptional programs in human pluripotent stem cells

“…Beads were washed twice with RIPA buffer, once with 2 M Urea in 10 mM Tris-HCl pH 8, twice with RIPA buffer and twice with PBS. Proteins were eluted from the beads in 50 μL elution buffer (200 mM NaCl, 50 mM Tris-HCl pH 8, 2% SDS, and 1 mM Biotin in water) for 30 min at 60 °C 51 . Protein samples were resolved by SDS–PAGE, transferred to nitrocellulose and probed with the indicated antibodies.…”

Co-targeting androgen receptor and DNA for imaging and molecular radiotherapy of prostate cancer: In vitro studies

“…DHT inhibits ∼70% of 125 IRISAD‐P binding in PC‐3 and ∼50% in DU 145 lysates. Estimated >110,000 AR copies (>1.64 × 10 −4 fmoles/cell) are detected in the LNCaP cell, significantly more than reported for the radioligand binding assay but in line with 127,000 ± 61,000 AR copies per cell determined in the quantitative mass spectrometry . PC‐3 cells have ∼25,000 AR/cell (3.90 × 10 −5 fmoles/cell) and DU 145 cells have ∼46,000 AR/cells (7.14 × 10 −5 fmoles/cell).…”