Gel filtration chromatography was performed on a TSK G300SB-C18 (7.5 × 600 mm, 10 μm, TOSOH) equilibrated with 50 mM Tris-HCl, 200 mM NaCl, pH 7.4. The column was connected to the Agilent 1100 series HPLC (Agilent, Santa Clara, CA) and eluted at a rate of 0.6 ml/min. Absorbance was read at 280 nm. The column was calibrated using the gel-filtration high–molecular weight kits (GE Healthcare Life Sciences, Little Chalfont, UK, and Sigma-Aldrich, St. Louis, MO). HPLC fractions were collected using the Agilent analytical fraction collector.
Selected fractions from the gel filtration column were concentrated by ultrafiltration (Mr > 10,000; Centricon YM-10; Amicon) and applied to HPLC mass spectrometry for mass determination of intact proteins. Proteins were separated by reverse-phase HPLC (Agilent 1100 series HPLC; Agilent) with a Zorbax 300SB-C18 (2.1 × 50 mm, 3.5 μm; Agilent) and introduced into the mass spectrometer as described (Apffel et al., 1995 ; Taggart et al., 2000 (link)). Positive-ion electrospray ionization mass spectra for intact proteins were obtained with an Agilent 6224 mass spectrometer equipped with an ion electrospray ionization interface and a time-of-flight mass detector (Agilent). Mass spectra were analyzed and deconvoluted as described (Stevens et al., 2009 (link)) using MassHunter, version B.04.00 (Agilent).
Selected fractions from the gel filtration column were concentrated by ultrafiltration (Mr > 10,000; Centricon YM-10; Amicon) and applied to HPLC mass spectrometry for mass determination of intact proteins. Proteins were separated by reverse-phase HPLC (Agilent 1100 series HPLC; Agilent) with a Zorbax 300SB-C18 (2.1 × 50 mm, 3.5 μm; Agilent) and introduced into the mass spectrometer as described (Apffel et al., 1995 ; Taggart et al., 2000 (link)). Positive-ion electrospray ionization mass spectra for intact proteins were obtained with an Agilent 6224 mass spectrometer equipped with an ion electrospray ionization interface and a time-of-flight mass detector (Agilent). Mass spectra were analyzed and deconvoluted as described (Stevens et al., 2009 (link)) using MassHunter, version B.04.00 (Agilent).