Evaluation of recombinant human interferon alpha-2b structure and stability by in-gel tryptic digestion, H/D exchange and mass spectrometry.
Stability and structure of recombinant interferon alpha-2b (rHuINF alpha-2b) was studied by mass spectrometry (MALDI-TOF and Q-TOF MS), chromatography (LC-UV-FLD-DAD, LC-MS) and CD spectroscopy. Besides analysis of the substance according to Ph. Eur. methods, two additional mass spectrometric methods were developed. The aim of both methods was to estimate structure-stability relationship connected to methionine oxidation or protein degradation. Preservation or degradation of protein structure was confirmed by H/D exchange in four separate experiments. Kinetics of deuterium incorporation into macromolecule was monitored over 2670 min. Isoforms of rHuINF alpha-2b were separated by 2D gel electrophoresis. In-gel digestion with trypsin and mass spectrometric analysis, performed on four separated isoforms at the mass corresponding to the mass of rHuINF alpha-2b with oxidized methionines, confirmed oxidation of all methionines to a different extent. Another four isoforms observed in 2D gel are most likely dimers of the same macromolecules with scrambled disulphide bridges. Oxidation and dimerisation are consequences of protein interaction with oxidizing reagents in polyacrilamide gel.
Cindrić
Mario
M
PLIVA, Research & Development Ltd., Prilaz Baruna Filipovića 29, 10000 Zagreb, Croatia. mario.cindric@pliva.hr
Galić
Nives
N
Vuletić
Marko
M
Klarić
Mia
M
Drevenkar
Vlasta
V
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002853:Chromatography, Liquid; D002942:Circular Dichroism; D004355:Drug Stability; D005782:Gels; D006801:Humans; D006868:Hydrolysis; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry*; D007700:Kinetics; D016014:Linear Models; D013058:Mass Spectrometry; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin
D005782:Gels; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins; D014357:Trypsin