Differential protein labeling with thiol-reactive infrared DY-680 and DY-780 maleimides and analysis by two-dimensional gel electrophoresis.
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 microg 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.
Riederer
Irène M
IM
Centre de Neurosciences Psychiatriques, Hôpital Psychiatrique, Prilly, Switzerland. Beat.Riederer@unil.ch
Riederer
Beat M
BM
D003720:Densitometry; D015180:Electrophoresis, Gel, Two-Dimensional; D008301:Maleimides / Q000737:chemistry*; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D012680:Sensitivity and Specificity; D019265:Spectroscopy, Near-Infrared; D013194:Staining and Labeling / Q000379:methods*; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D008301:Maleimides; D011506:Proteins; D013438:Sulfhydryl Compounds