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Differential protein labeling with thiol‐reactive infrared DY‐680 and DY‐780 maleimides and analysis by two‐dimensional gel electrophoresis
Author(s) -
Riederer Irène M.,
Riederer Beat M.
Publication year - 2007
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200601007
Subject(s) - chemistry , cysteine , thiol , maleimide , gel electrophoresis , polyacrylamide gel electrophoresis , biochemistry , chromatography , blot , organic chemistry , enzyme , gene
Differential protein labeling with 2‐DE separation is an effective method for distinguishing differences in the protein composition of two or more protein samples. Here, we report on a sensitive infrared‐based labeling procedure, adding a novel tool to the many labeling possibilities. Defined amounts of newborn and adult mouse brain proteins and tubulin were exposed to maleimide‐conjugated infrared dyes DY‐680 and DY‐780 followed by 1‐ and 2‐DE. The procedure allows amounts of less than 5 µg of cysteine‐labeled protein mixtures to be detected (together with unlabeled proteins) in a single 2‐DE step with an LOD of individual proteins in the femtogram range; however, co‐migration of unlabeled proteins and subsequent general protein stains are necessary for a precise comparison. Nevertheless, the most abundant thiol‐labeled proteins, such as tubulin, were identified by MS, with cysteine‐containing peptides influencing the accuracy of the identification score. Unfortunately, some infrared‐labeled proteins were no longer detectable by Western blots. In conclusion, differential thiol labeling with infrared dyes provides an additional tool for detection of low‐abundant cysteine‐containing proteins and for rapid identification of differences in the protein composition of two sets of protein samples.