Method for qualitative comparisons of protein mixtures based on enzyme-catalyzed stable-isotope incorporation.
Determining which proteins are unique among one or several protein populations is an often-encountered task in proteomics. To this purpose, we present a new method based on trypsin-catalyzed incorporation of the stabile isotope (18)O in the C-termini of tryptic peptides, followed by LC-MALDI MS analysis. The analytical strategy was designed such that proteins unique to a given population out of several can be assigned in a single experiment by the isotopic signal intensity distributions of their tryptic peptides in the recorded mass spectra. The method is demonstrated for protein-protein interaction analysis, in which the differential isotope labeling was used to distinguish endogenous human brain proteins interacting with a recombinant bait protein from nonbiospecific background binders.
Mirgorodskaya
Ekaterina
E
Max Planck Institute for Molecular Genetics, Berlin, Germany.
Wanker
Erich
E
Otto
Albrecht
A
Lehrach
Hans
H
Gobom
Johan
J
D001921:Brain / Q000378:metabolism; D002384:Catalysis; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D047369:Chromatin Immunoprecipitation; D002853:Chromatography, Liquid / Q000379:methods*; D030562:Databases, Protein; D004591:Electrophoresis, Polyacrylamide Gel; D004798:Enzymes / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D010100:Oxygen / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000737:chemistry / Q000494:pharmacology
D004798:Enzymes; D010455:Peptides; D011506:Proteins; D020543:Proteome; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D014357:Trypsin; D010100:Oxygen