Journal: Scientific Reports

Article Title: Quantitative proteomics of Xenopus laevis embryos: expression kinetics of nearly 4000 proteins during early development

doi: 10.1038/srep04365

Figure Lengend Snippet: Micrographs of Xenopus laevis embryos at developmental stages used for iTRAQ measurements (A). Design and workflow of three independent iTRAQ experiments (B). Three experiments were performed. In experiment 1 (E1), two embryos at stage 1, two embryos at stage 5, two embryos at stage 8, and two embryos at stage 11 were separately lysed and digested with trypsin. The first embryo at stage 1 was labeled with the iTRAQ reagent channel 113, the second embryo at stage 1 was labeled with the iTRAQ reagent channel 114, the two embryos at stage 5 were labeled with the iTRAQ reagents channels 115 and 116, the two embryos at stage 8 were labeled with the iTRAQ reagent channels 117 and 118, and the two embryos at stage 11 were labeled with the iTRAQ reagent channels 119 and 121. These labeled peptides were pooled, subjected to strong cation exchange chromatography fractionation, and each fraction was analyzed using reversed-phase liquid chromatography and detection with a Q-Exactive mass spectrometer. Tandem mass spectra were analyzed both to identify the peptide and to quantitate the abundances of each peptide from each of the eight embryos. A similar procedure was performed in experiment 2 (E2), except that the biological duplicates consisted of single embryos taken from stages 1, 5, 13 and 22. Finally, experiment 3 (E3) employed four pools of four embryos, where each pool was taken embryos at stages 1, 8, 13 and 22.

Article Snippet: Following SDS-PAGE, proteins were transferred to a nitrocellulose membrane overnight at 4°C and developed with antibody, produced in rabbit, specific to Xenopus laevis VgRBP71 (generated in the Huber laboratory), actin (AHP1629, AbD Serotec, Raleigh, NC) and Cdc6 (kindly provided by the laboratory of Dr. William G. Dunphy at the California Institute of Technology).

Techniques: Multiplex sample analysis, Labeling, Chromatography, Fractionation, Reversed-phase Chromatography, Mass Spectrometry