Proteomic analysis of changes during the onset of amphibian limb regeneration

Abstract

Synthesis of specific proteins has been examined during the early phase of limb regeneration, but an overall analysis of protein changes in this period has been lacking. We report the first global proteomic analysis of this system, utilizing LC/MS‐based identification and quantification methods to compare abundant proteins in hindlimbs of the larval anuran Xenopus laevis (stage 53) at the time of amputation (0dPA) and 3 days later when the regeneration blastema is developing (3dPA). We identified and quantified 1517 peptides, of which 1067 were identified with high peptide ID confidence (555 with multiple peptides and 512 with a single peptide). Of these 1067 proteins, 489 showed significant changes in quantity between the two groups. Among those proteins that were elevated in the tissue relative to 0dPA were members of the annexin family (e.g. ANXA1, ANXA2, ANXA5) and the ANXA2‐binding partner S100A10, which have important immunoregulatory roles in other systems and which were also shown to be differentially expressed in stage 53 and 57 3dPA and 5dPA tissues in our previous microarray studies. Quantitative PCR (qPCR) revealed that the differences in annexins and S100A10 protein levels were consistent with the differences in levels of gene expression. Besides elucidating the possible modulation of inflammation during amphibian limb regeneration, the results also provide insight into the dedifferentiation process by revealing up‐regulation of various proteins known to characterize many stem cells. This work was supported by a pilot grant from the Indiana University School of Medicine.