Modified electrophoretic and digestion conditions allow a simplified mass spectrometric evaluation of disulfide bonds.
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.
Pompach
Petr
P
Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
Man
Petr
P
Kavan
Daniel
D
Hofbauerová
Katerina
K
Kumar
Vinay
V
Bezouska
Karel
K
Havlícek
Vladimír
V
Novák
Petr
P
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*
D004220:Disulfides; D011506:Proteins