Modified electrophoretic and digestion conditions allow a simplified mass spectrometric evaluation of disulfide bonds
Language English Country England, Great Britain Media print
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
19551705
DOI
10.1002/jms.1609
Knihovny.cz E-resources
- MeSH
- Disulfides chemistry MeSH
- Electrophoresis, Polyacrylamide Gel methods MeSH
- Mass Spectrometry methods MeSH
- Humans MeSH
- Molecular Sequence Data MeSH
- Proteins chemistry MeSH
- Amino Acid Sequence MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Disulfides MeSH
- Proteins MeSH
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.
References provided by Crossref.org
Natural Killer Cell Activation Receptor NKp30 Oligomerization Depends on Its N-Glycosylation
Production of recombinant soluble dimeric C-type lectin-like receptors of rat natural killer cells
Oligomeric Architecture of Mouse Activating Nkrp1 Receptors on Living Cells
Crystallization and diffraction analysis of β-N-acetylhexosaminidase from Aspergillus oryzae
