Mayor, T., Lipford, J.R., Graumann, J., Smith, G.T. and Deshaies, R.J.
Notes: These authors used a two-step process to purify a spectrum of ubiquitylated proteins from yeast expressing polyhistidine-tagged ubiquitin. First, two polyubiquitin-binding proteins were fused to glutathione-S-transferase purification tags (GST-Rad23 and GST-Dsk2), expressed in bacteria and purified by glutathione-Sepharose resin. A cleared yeast lysate was added to the GST-coupled proteins, mixed and washed. Elution of bound proteins was performed using a urea buffer (8M urea, 100mM NaH2PO4, 10mM Tris-HCl, pH 8.0). The eluate was then mixed with 125µl MagneHis™ Ni-Particles previously washed in the urea buffer. After incubation, the MagneHis™ particles were stringently rinsed with urea buffer + 0.5% Triton® X-100. To generate peptides for MS-based sequencing, proteolytic digests were performed directly on the beads. The enzymes used were endoproteinase Lys-C (incubated for 5 hours) and trypsin (incubated for 16 hours). After digestion, the protein sample was analyzed by mass spectrometry (ESI-MS). (3286)