A comparative proteome and phosphoproteome analysis of differentially regulated proteins during fertilization in the self-incompatible species Solanum chacoense Bitt

We have used 2‐DE for a time‐course study of the changes in protein and phosphoprotein expression that occur immediately after fertilization in Solanum chacoense. The phosphorylation status of the detected proteins was determined with three methods: in vivo labeling, immunodetection, and phosphoprotein‐specific staining. Using a pI range of 4–7, 262 phosphorylated proteins could be mapped to the 619 proteins detected by Sypro Ruby staining, representing 42% of the total proteins. Among these phosphoproteins, antibodies detected 184 proteins from which 78 were also detected with either of the other two methods (42%). Pro‐Q Diamond phosphoprotein stain detected 111 proteins, of which 76 were also detected with either of the other two methods (68%). The 32P in vivo labeling method detected 90 spots from which 78 were also detected with either of other two methods (87%). On comparing before and after fertilization profiles, 38 proteins and phosphoproteins presented a reproducible change in their accumulation profiles. Among these, 24 spots were selected and analyzed by LC‐MS/MS using a hybrid quadrupole‐TOF (Q‐TOF) instrument. Peptide data were searched against publicly available protein and EST databases, and the putative roles of the identified proteins in early fertilization events are discussed.