Identification of Binding Proteins in Xenopus laevis by MALDI-TOF/TOF Mass Spectrometry

Abstract

MALDI-TOF/TOF MS has been widely used for the identification of proteins in the proteomics field. Here we tried to identify novel proteins during vertebrate development by this MS and targeted the protein binding to Xenopus β-arrestin 2 (xβarr2). First, we prepared total lysate from dorsal marginal zone (DMZ) tissues of Xenopus gastrula embryos. To isolate proteins from DMZ lysate, which bind with xβarr2, we performed GST pulldown assay with GST-fused xβarr2 protein and then separated the proteins by SDS-PAGE. Finally, the bound proteins were analyzed in the tandem MS (TOF/TOF) mode to generate fragment ions for determination of their amino acid sequence. As the result, we identified Xenopus β-tubulin (xβTub) as a binding partner of xβarr2. To verify this discovering method, we performed several in vivo and in vitro experiments. Whole mount in situ hybridization showed that xβTub had similar expression pattern to that of xβarr2 during Xenopus gastrulation. Moreover, forced expression of xβTub caused severe gastrulation cell movements, which is previously shown in xβarr2 overexpression. Protein-protein interaction of xβarr2 and xβTub was also verified by immunoprecipitation, suggesting that we have demonstrated that discovering binding partner of a certain protein can be successfully done by MALDI-TOF/TOF MS in Xenopus embryonic system.

Keywords

• MALDI-TOF/TOF
• Mass spectrometry
• β-tubulin
• β-arrestin 2
• gastrulation
• Xenopus