Naslov
Evaluation of Recombinant Human Interferon a-2b Structure and Stability by In-gel Tryptic Digestion, H/D Exchange and Mass Spectrometry

Autori
Cindrić, Mario ; Galić, Nives ; Vuletić, Marko ; Klarić, Mia ; Drevenkar, Vlasta

Izvornik
Journal of pharmaceutical and biomedical analysis (0731-7085) 40 (2006), 3; 781-787

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
recombinant human interferon a-2b; stability; MALDI-TOF MS; H/D exchange; 2-D gel electrophoresis; methionine oxidation

Sažetak
Stability and structure of recombinant interferon a-2b (rHuINF a-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. The kinetics of deuterium incorporation into macromolecule was monitored over 2670 minutes. Isoforms of rHuINF a-2b were separated by 2-D 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 a-2b with oxidized methionines, confirmed oxidation of all methionines to a different extent. Another four isoforms observed in 2-D 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.

Izvorni jezik
Engleski

Znanstvena područja
Kemija

Napomena
S.I: Hyphenated Techniques in Pharmaceutical and Biomedical Analysis ; B. Chankvetadze (ur.).

POVEZANOST RADA