Comparative proteomic approaches for the isolation of proteins interacting with thioredoxin.
Thioredoxin (TRX) is a small multifunctional protein with a disulfide active site involved in redox regulation. To gain insight into the numerous proteins able to interact with thioredoxin in Arabidopsis thaliana, we have compared three different proteomic procedures. In the two first approaches targets present in a mixture of soluble leaf proteins were reduced by the cytosolic TRX h3, then the new thiols were labeled either with radioactive iodoacetamide allowing specific detection (first method) or with a biotinylated thiol-specific compound allowing selective retention on an avidin column (second method). The third method involved a chromatography on a mutated TRX h3 column, which is able to covalently trap potential targets. All together, the three approaches enabled us to propose 73 proteins as being TRX-linked, and involved in various processes. Methods 1 and 3 were not only efficient with respectively 47 and 41 potential targets, but also complementary as only 26% of the targets were identified by both procedures. The second method with only 12 proteins was less efficient. However, this approach, as well as the first one when coupled with differential labeling of the cysteine residues, could be more informative about the cysteines involved in the thiol-disulfide interchange.
Marchand
Christophe
C
IBBMC, CNRS UMR 8619, Université Paris-Sud, Orsay, France.
Le Maréchal
Pierre
P
Meyer
Yves
Y
Decottignies
Paulette
P
D017360:Arabidopsis / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D001710:Biotin; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D007457:Iodine Radioisotopes; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011485:Protein Binding; D025941:Protein Interaction Mapping; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*
D029681:Arabidopsis Proteins; D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D013879:Thioredoxins; D001710:Biotin