Exploration of phosphoproteomic association during epimorphic regeneration

Abstract

Unravelling the intricate patterns of site-specific protein phosphorylation during Epimorphic regeneration holds the key to unlocking the secrets of tissue complexity. Understanding these precise modifications and their impact on protein function could shed light on the remarkable regenerative capacity of tissues, with potential implications for therapeutic interventions. In this study we have systematically mapped the global phosphorylation modifications within regenerating tissue of zebrafish caudal fins, elucidating the intricate landscape of signalling pathway associate with the regeneration process. Based on mass spectrometry analysis, we identified 440 phosphorylated proteins using the immunoprecipitation method with phosphoserine, phosphothreonine, and phosphotyrosine antibodies, and 74 phosphorylated proteins using the TiO₂ column enrichment method were found differentially phosphorylated during the regeneration process from 12 hpa to 7 dpa compared to the control. Interestingly 95% of the proteins identified from TiO₂ enrichment method were also found to be identified through the phosphoprotein antibody pull down method impacting the high accuracy and significance of the methods and greater association of the 70 proteins undergoing differential phosphorylation during the process of regeneration. Whole mount immunohistochemistry analysis reveals high association of phosphorylation at 1dpa, 2dpa and 3dpa regeneration time points. Network pathway analysis revealed that cancer-related diseases, organismal injuries and abnormalities as the most strongly associated canonical network pathways with the differentially expressed phosphoproteome in the mechanism of regeneration. This research enhances our comprehension on protein post-translational modification in the context of zebrafish caudal fin tissue regeneration, shedding light on its prospective application in the field of regenerative medicine.

Keywords: Immunoprecipitation; Phosphoprotein; Proteomics; Regeneration; TiO2; Zebrafish.

Fig. 1

Heat map analysis and Graphical Representation of fold changes of differentially phosphorylated proteins in regenerating zebrafish caudal fin tissue relative to non-regenerating tissue, (a) Phosphoproteome heat map analysis and (b) Expression pattern analysis of phosphoproteins relative to control represented as the mean ± SEM.

Fig. 2

Wholemount immunohistochemistry analysis of differential phosphorylation associated with zebrafish caudal fin regeneration and expression plot at each timepoints with respect to phosphoserine, phosphothreonine and phosphotyrosine antibodies respectively.

Fig. 3

Western blot analysis depicting phosphoproteins for the antibodies at different timepoints 0hpa, 12hpa, 1dpa, 2dpa, 3dpa and 7dpa and their relative quantification against 0hpa. (a) blot with phosphoserine antibody (b) blot with phosphothreonine antibody, and (c) blot with phosphotyrosine antibody.

Fig. 4

Canonical network pathways and disease & biofunctions pathways associated with the differentially expressed phosphoproteome for zebrafish caudal fin regeneration (a) Heat map of top 15 canonical pathways. (b) Heat map of disease and bio functions. (c) Cancer, haematological & immunological disease canonical pathway (d) Cancer, organismal injury & abnormalities canonical network pathways.