Post-translational modification by SUMO is a highly conserved pathway in 
eukaryotes that plays very important regulatory roles in many cellular 
processes. Deregulation of the SUMO pathway contributes to the development and 
progression of many diseases including cancer. Therefore, identifying additional 
SUMO substrates and studying how their cellular and biological functions are 
regulated by sumoylation should provide new insights. Our studies showed that 
sumoylation activity was significant in Xenopus egg extracts, and that a high 
level of sumoylation was associated with sperm chromatin when SUMO was incubated 
with Xenopus egg extracts. By isolating SUMO-conjugated substrates using 
His-tagged SUMO1 or SUMO2 proteins under denaturing conditions, we identified 
346 proteins by mass spectrometry analysis that were not present in control 
pull-downs. Among them, 167 proteins were identified from interphase egg 
extracts, 86 proteins from mitotic phase egg extracts, and 93 proteins from 
both. Thirty-three proteins were pulled down by SUMO1, 85 proteins by SUMO2, and 
228 proteins by both. We validated the sumoylation of five candidates, CKB, 
ATXN10, BTF3, HABP4, and BZW1, by co-transfecting them along with SUMO in 
HEK293T cells. Gene ontology analysis showed that SUMO substrates identified in 
this study were involved in diverse biological processes. Additionally, SUMO 
substrates identified from different cell cycle stages or pulled down by 
different SUMO homologs were enriched for distinct cellular components and 
functional categories. Our results comprehensively profile the sumoylation 
occurring in the Xenopus egg extract system.