HyperISGylation of Old World Monkey ISG15 in Human Cells

Pattyn, Els; Verhee, Annick; Uyttendaele, Isabel; Piessevaux, Julie; Timmerman, Evy; Gevaert, Kris; Vandekerckhove, Joël; Peelman, Frank; Tavernier, Jan

The 2fTGH or Hek293T cells expressing either TAP-tagged AgmISG15 or HuISG15 were lysed in cell lysis buffer (50 mM Tris-HCl pH 8, 10% glycerol, 1% NP40, 150 mM NaCl, 5 mM NaF, 5 mM ZnCl 2 , 1 mM Na 3 VO 4 , 10 mM EGTA, Complete TM Protease Inhibitor Cocktail (Roche)). The insoluble fraction was spun down and the supernatant was incubated with IgG sepharose (Amersham Biosciences) overnight. The beads were washed three times with washing buffer (2 mM Tris-HCl pH 7.5, 5% glycerol, 0.1% NP40, 150 mM NaCl) and twice with TEV (Tobacco Etch Virus) protease cleavage buffer 1 (10 mM Tris-HCl pH 8, 150 mM NaCl, 0.1% NP40, 0.5 mM EDTA) and were then incubated with TEV protease in TEV protease cleavage buffer 1 for 2 hours. The beads were then spun down and the supernatant was incubated with anti-FLAG agarose (Sigma) in TEV protease cleavage buffer 2 (10 mM Tris-HCl pH 8, 150 mM NaCl, 0.1% NP40) for 2 to 4 hours. The anti-FLAG agarose beads were washed three times with washing buffer and incubated with 250 mg/mL FLAG peptide in FLAG elution buffer (2 mM Tris-HCl pH 7.5, 5% glycerol, 150 mM NaCl) for 10 min to elute for the FLAG-tagged proteins. The resulting peptide mixture was precipitated by addition of TCA to a final concentration of 10%. It was incubated overnight, centrifuged and washed with ice-cold acetone containing 0.05N HCl and dried. Pellets were re-dissolved in 3 mL 50 mM NH 4 HCO 3 (pH 7.9) containing 8M ureum for 20min periodic vortexing, 21 mL 50 mM NH 4 HCO 3 (pH 7.9) was added in aliquots to lower the final concentration of ureum to 1 M. The resulting peptide mix was digested in solution by trypsin and applied for nano-LC-MS/MS analysis as described before using a Waters Q-TOF mass spectrometer [58] or a Bruker Esquire HCT ion trap mass spectrometer [59] .