Proteomic analysis of the androgen receptor via MS-compatible purification of biotinylated protein on streptavidin resin

• Published in: Proteomics (Weinheim) Vol. 12; no. 1; pp. 43 - 53
• Main Authors: Austin, Ryan J., Smidansky, Heidi M., Holstein, Carly A., Chang, Deborah K., Epp, Angela, Josephson, Neil C., Martin, Daniel B.
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 01.01.2012; WILEY‐VCH Verlag; Wiley-VCH; Wiley Subscription Services, Inc
• Subjects: Absolute SILAC; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Androgen receptor; Androgens; Biological and medical sciences; Biotin - isolation & purification; Biotin - metabolism; Biotinylation; Cell biology; Cell Line, Tumor; Chromatography, Affinity - methods; Fundamental and applied biological sciences. Psychology; Gynecology. Andrology. Obstetrics; Humans; Male genital diseases; Mass Spectrometry - standards; Medical sciences; Miscellaneous; Molecular Sequence Data; Nephrology. Urinary tract diseases; Peptide Fragments - chemistry; Prostate cancer; Protein Binding; Protein Interaction Mapping - methods; Proteins; Proteomics; Receptors, Androgen - biosynthesis; Receptors, Androgen - isolation & purification; Receptors, Androgen - metabolism; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; Reference Standards; Saccharomyces cerevisiae; SILAC; Streptavidin; Streptavidin - chemistry; Tumors; Tumors of the urinary system; Urinary tract. Prostate gland; Urinary system disease; Purification; Prostate disease; Androgen; Cell biology; Malignant tumor; SILAC; Protein; Absolute SILAC; Proteomics; Androgen receptor; Streptavidin; Male genital diseases; Prostate cancer; Cancer
• ISSN: 1615-9853; 1615-9861
• DOI: 10.1002/pmic.201100348

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.