title: Modified electrophoretic and digestion conditions allow a simplified mass spectrometric evaluation of disulfide bonds.
abstract: Proper formation of disulfide bonds in proteins is a prerequisite to their stability and function. Information on disulfide pattern may therefore serve as an indication of the proper folding of recombinant proteins, and can also be used in protein homology modeling for the purpose of structure refinement. Protein handling and digestion at basic pH leads to disulfide bond scrambling. That is why the samples are usually treated and digested at low pH where no scrambling occurs. Unfortunately, the specific proteases used in protein research are active at high pH values. Here, we present a complete sample handling protocol, which allows processing of disulfide containing proteins at basic pH. We modified the standard SDS gel electrophoresis and protein digestion conditions by the addition of an oxidative agent, cystamine. This modification prevented disulfide scrambling, which we otherwise observed in the samples handled according to the general protocol. Lysozyme from hen egg was used as a model protein for the development of the method. We then applied our protocol to human leukocyte antigen CD69, for which the disulfide bonding is known, but only for its monomeric form. In addition, the disulfide arrangement was then 'de novo' identified in the recombinant murine leukocyte receptor NKR-P1A and in the larger glycosylated proteins beta-N-acetylhexosaminidases from Aspergillus oryzae and Penicillium oxalicum.
authors: Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors: Petr
authors: P
authors: Pompach
authors: Petr
authors: P
authors: Man
authors: Daniel
authors: D
authors: Kavan
authors: Katerina
authors: K
authors: Hofbauerová
authors: Vinay
authors: V
authors: Kumar
authors: Karel
authors: K
authors: Bezouska
authors: Vladimír
authors: V
authors: Havlícek
authors: Petr
authors: P
authors: Novák
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004220:Disulfides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D011506:Proteins / Q000737:chemistry*
chemical_list: D004220:Disulfides; D011506:Proteins