Comparative Peptidomics Analysis Of Hydrogen Peroxide-Induced Apoptosis In Saccharomyces Cerevisiae

Peptidomics have been applied to detect peptides derived from intracellular proteins using mass spectrometry-based techniques.The applications of peptidomics are involved in identifying biomarker,detecting peptidases and proteases substrates,and elucidating the proteolytic pathways.Apoptosis is a process of programmed cell death,which is highly regulated by genes.It is essential for the development and maintenance of homeostasis of multicellular organisms.Reactive oxygen species?ROS?are key regulator of apoptosis,little is known about ROS how to regulate apoptosis.To date,Yca1p is the only mammalian homologue of mammalian caspase protein found in yeast.YCA1 plays an important role in the regulation of yeast apoptosis,but its molecular mechanism remains to be further studied.In order to further study the mechanism of reactive oxygen species?ROS?and YCA1in apoptosis,we used BY4741 and?yca1 as experimental materials,and hydrogen peroxide as oxidative stress.Different concentrations of hydrogen peroxide were applied to treat BY4741 and?yca1,respectively.Cell viability was assesed by counting the colony forming units,and Annexin V/PI costaining were used to quantify the rate of apoptosis,respectively.Using quantitative peptidomics technique to investigate the peptidomics response of yeast cell to H₂O₂,further elaborates the mechanism of ROS in yeast apoptosis.By comparing the changes of peptides in both strains,find out the molecular mechanism of YCA1 in the process of apoptosis.The experimental results are as follows:1.Colony forming units and Annexin V-FITC/PI double staining showed that when using different concentrations of hydrogen peroxide treatment of wild type BY4741 and mutant strains?yca1,with the increase of concentration,the survival rate decreased.In contrast,apoptosis rate increased gradually.The survival rate of?yca1was higher than that of wild type BY4741,and the apoptosis rate was lower than that of wild type BY4741.2.Wild type BY4741 were treated with 2 mM H₂O₂,a total of 912 distinct peptides were detected.There were significant changes in 262 peptides,which derived from 121proteins,48%of the differentially expressed peptides were detected in the N or C terminal of the precursor protein.Glyceraldehyde-3-phosphate dehydrogenase 3,phosphoglycerate kinase,fructose-bisphosphate aldolase,60S acidic ribosomal protein P1-alpha and heat shock protein 60 can produce more than 6 peptides.Peptides derived from these 5 proteins were up-regulated and down-regulated.3.A total of 490 peptides were identified by 2 mM H₂O₂ treat?yca1.There were significant changes in 170 peptides,which belonging to 87 precursor proteins.Among them,two up-regulated peptides gQDDLGkGDTEES and tPGAATIkPTVE were derived from superoxide dismutase[Cu-Zn]and peroxidase TSA1.50%differentially expressed peptides were located in the N or C terminal of the precursor protein.Most of the peptides derived from characterized protein YNL208W,phosphoglycerate kinase,elongation factor1-alpha,and co-chaperone protein SBA-1 degradation.4.By comparison of the peptides of wild type BY4741 and mutant strains?yca1 with2 mM H₂O₂ treatment,found that 300 identical peptides,of which 182 peptides changes significantly.the changes were observed in two strains are 42 peptides,only significant changes in the wild-type BY4741 is 45 peptides,only significant changes in the mutant?yca1 is 95 peptides.5.It was further demonstrated that GAPDH is a specific cleavage substrate of YCA1and STM1 is also likely to be a substrate of YCA1.In this study,we investigated the mechanism of apoptosis and the role of YCA1 by quantitative peptidomics,which is the first time to study the molecular mechanism of apoptosis in peptide level.In our research,we found many differentially expressed peptides during apoptosis,which may play some roles in apoptosis.The next step is functional verification.